Systems for treating pulmonary infections

ABSTRACT

Provided herein are systems for treating a subject with a pulmonary infection, for example, a nontuberculous mycobacterial pulmonary infection, a  Burkholderia  pulmonary infection, a pulmonary infection associated with bronchiectasis, or a  Pseudomonas  pulmonary infection. The system includes a pharmaceutical formulation comprising a liposomal aminoglycoside dispersion, and the lipid component of the liposomes consist essentially of electrically neutral lipids. The system also includes a nebulizer which generates an aerosol of the pharmaceutical formulation at a rate greater than about 0.53 gram per minute. The aerosol is delivered to the subject via inhalation for the treatment of the pulmonary infection.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/919,771, filed Jul. 2, 2020, which is a continuation of U.S.application Ser. No. 16/682,674, filed Nov. 13, 2019, now abandoned,which is a continuation of U.S. application Ser. No. 16/010,094, filedJun. 15, 2018, now abandoned, which is a continuation of U.S.application Ser. No. 15/900,261, filed Feb. 20, 2018, now abandoned,which is a continuation of U.S. application Ser. No. 15/379,804, filedDec. 15, 2016, now abandoned, which is a continuation of U.S.application Ser. No. 13/899,457, filed May 21, 2013, now U.S. Pat. No.9,566,234, which claims the benefit of priority from U.S. ProvisionalApplication No. 61/649,830, filed May 21, 2012, each of which is herebyincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Certain technologies suitable for administration by inhalation employliposomes and lipid complexes supply a prolonged therapeutic effect ofdrug in the lung. These technologies also provide the drug withsustained activities, and the ability to target and enhance the uptakeof the drug into sites of disease.

Inhalation delivery of liposomes is complicated by their sensitivity toshear-induced stress during nebulization, which can lead to change inphysical characteristics (e.g., entrapment, size). However, as long asthe changes in characteristics are reproducible and meet acceptabilitycriteria, they need not be prohibitive to pharmaceutical development.

Cystic fibrosis (CF) patients have thick mucus and/or sputum secretionsin the lungs, frequent consequential infections, and biofilms resultingfrom bacterial colonizations. All these fluids and materials createbarriers to effectively targeting infections with aminoglycosides.Liposomal aminoglycoside formulations may be useful in combating thebacterial biofilms.

SUMMARY OF THE INVENTION

The present invention provides methods for treating various pulmonaryinfections, including mycobacterial infections (e.g., pulmonaryinfections caused by nontuberculous mycobacterium, also referred toherein as nontuberculous mycobacterial (NTM) infections), by providingsystems for delivery of aerosolized liposomal formulations viainhalation. For example, the systems and methods provided herein can beused to treat a pulmonary nontuberculous mycobacterial infection such aspulmonary M. avium, M. avium subsp. hominissuis (MAH), M. abscessus, M.chelonae, M. bolletii, M. kansasii, M. ulcerans, M. avium, M. aviumcomplex (MAC) (M. avium and M. intracellulare), M. conspicuum, M.kansasii, M. peregrinum, M. immunogenum, M. xenopi, M. marinum, M.malmoense, M. marinum, M. mucogenicum, M. nonchromogenicum, M.scrofulaceum, M. simiae, M. smegmatis, M. szulgai, M. terrae, M. terraecomplex, M. haemophilum, M. genavense, M. gordonae, M. ulcerans, M.fortuitum or M. fortuitum complex (M. fortuitum and M. chelonae)infection.

In one aspect, the present invention provides a system for treating orproviding prophylaxis against a pulmonary infection. In one embodiment,the system comprises a pharmaceutical formulation comprising a liposomalcomplexed aminoglycoside, wherein the formulation is a dispersion (e.g.,a liposomal solution or suspension), the lipid component of the liposomeconsists of electrically neutral lipids, and a nebulizer which generatesan aerosol of the pharmaceutical formulation at a rate greater thanabout 0.53 g per minute. In one embodiment, the mass median aerodynamicdiameter (MMAD) of the aerosol is less than about 4.2 μm, as measured bythe Anderson Cascade Impactor (ACI), about 3.2 μm to about 4.2 μm, asmeasured by the ACI, or less than about 4.9 μm, as measured by the NextGeneration Impactor (NGI), or about 4.4 μm to about 4.9 μm, as measuredby the NGI.

In another embodiment, the system for treating or providing prophylaxisagainst a pulmonary infection comprises a pharmaceutical formulationcomprising a liposomal complexed aminoglycoside, wherein the formulationis a dispersion (e.g., a liposomal solution or suspension), the lipidcomponent of the liposome consists of electrically neutral lipids, and anebulizer which generates an aerosol of the pharmaceutical formulationat a rate greater than about 0.53 g per minute. The fine particlefraction (FPF) of the aerosol is greater than or equal to about 64%, asmeasured by the Anderson Cascade Impactor (ACI), or greater than orequal to about 51%, as measured by the Next Generation Impactor (NGI).

In one embodiment, the system provided herein comprises a pharmaceuticalformulation comprising an aminoglycoside. In a further embodiment, theaminoglycoside is amikacin, apramycin, arbekacin, astromicin,capreomycin, dibekacin, framycetin, gentamicin, hygromycin B,isepamicin, kanamycin, neomycin, netilmicin, paromomycin,rhodestreptomycin, ribostamycin, sisomicin, spectinomycin, streptomycin,tobramycin, verdamicin or a combination thereof. In even a furtherembodiment, the aminoglycoside is amikacin. In another embodiment, theaminoglycoside is selected from an aminoglycoside set forth in Table A,below, or a combination thereof.

TABLE A AC4437 amikacin apramycin arbekacin astromicin bekanamycinboholmycin brulamycin capreomycin dibekacin dactimicin etimicinframycetin gentamicin H107 hygromycin hygromycin B inosamycin K-4619isepamicin KA-5685 kanamycin neomycin netilmicin paromomycin plazomicinribostamycin sisomicin rhodestreptomycin sorbistin spectinomycinsporaricin streptomycin tobramycin verdamicin vertilmicin

The pharmaceutical formulations provided herein are dispersions ofliposomes (i.e., liposomal dispersions or aqueous liposomal dispersionswhich can be either liposomal solutions or liposomal suspensions). Inone embodiment, the lipid component of the liposomes consistsessentially of one or more electrically neutral lipids. In a furtherembodiment, the electrically neutral lipid comprises a phospholipid anda sterol. In a further embodiment, the phospholipid isdipalmitoylphosphatidylcholine (DPPC) and the sterol is cholesterol.

In one embodiment, the lipid to drug ratio in the aminoglycosidepharmaceutical formulation (aminoglycoside liposomal solution orsuspension) is about 2:1, about 2:1 or less, about 1:1, about 1:1 orless, or about 0.7:1.

In one embodiment, the aerosolized aminoglycoside formulation, uponnebulization, has an aerosol droplet size of about 1 μm to about 3.8 μm,about 1.0 μm to 4.8 μm, about 3.8 μm to about 4.8 μm, or about 4.0 μm toabout 4.5 μm. In a further embodiment, the aminoglycoside is amikacin.In even a further embodiment, the amikacin is amikacin sulfate.

In one embodiment, about 70% to about 100% of the aminoglycoside presentin the formulation is liposomal complexed, e.g., encapsulated in aplurality of liposomes, prior to nebulization. In a further embodiment,the aminoglycoside is selected from an aminoglycoside provided in TableA. In further embodiment, the aminoglycoside is an amikacin. In even afurther embodiment, about 80% to about 100% of the amikacin is liposomalcomplexed, or about 80% to about 100% of the amikacin is encapsulated ina plurality of liposomes. In another embodiment, prior to nebulization,about 80% to about 100%, about 80% to about 99%, about 90% to about100%, 90% to about 99%, or about 95% to about 99% of the aminoglycosidepresent in the formulation is liposomal complexed prior to nebulization.

In one embodiment, the percent liposomal complexed (also referred toherein as “liposomal associated”) aminoglycoside post-nebulization isfrom about 50% to about 80%, from about 50% to about 75%, from about 50%to about 70%, from about 55% to about 75%, or from about 60% to about70%. In a further embodiment, the aminoglycoside is selected from anaminoglycoside provided in Table A. In a further embodiment, theaminoglycoside is amikacin. In even a further embodiment, the amikacinis amikacin sulfate.

In another aspect, the present invention provides methods for treatingor providing prophylaxis against a pulmonary infection. In oneembodiment, the pulmonary infection is a pulmonary infection caused by agram negative bacterium (also referred to herein as a gram negativebacterial infection). In one embodiment, the pulmonary infection is aPseudomonas infection, e.g., a Pseudomonas aeruginosa infection. Inanother embodiment, the pulmonary infection is caused by one of thePseudomonas species provided in Table B, below. In one embodiment, apatient is treated for mycobacterial lung infection with one of thesystems provided herein. In a further embodiment, the mycobacterialpulmonary infection is a nontuberculous mycobacterial pulmonaryinfection, a Mycobacterium abscessus pulmonary infection or aMycobacterium avium complex pulmonary infection. In one or more of thepreceding embodiments, the patient is a cystic fibrosis patient.

In one embodiment, a patient with cystic fibrosis is treated for apulmonary infection with one of the systems provided herein. In afurther embodiment, the pulmonary infection is caused by Mycobacteriumabscessus, Mycobacterium avium complex, or P. aeruginosa. In anotherembodiment, the pulmonary infection is caused by a nontuberculousmycobacterium selected from M. avium, M. avium subsp. hominissuis (MAH),M. abscessus, M. chelonae, M. bolletii, M. kansasii, M. ulcerans, M.avium, M. avium complex (MAC) (M. avium and M. intracellulare), M.conspicuum, M. kansasii, M. peregrinum, M. immunogenum, M. xenopi, M.marinum, M. malmoense, M. marinum, M. mucogenicum, M. nonchromogenicum,M. scrofulaceum, M. simiae, M. smegmatis, M. szulgai, M. terrae, M.terrae complex, M. haemophilum, M. genavense, M. asiaticum, M.shimoidei, M. gordonae, M. nonchromogenicum, M. triplex, M. lentiflavum,M. celatum, M. fortuitum, M. fortuitum complex (M. fortuitum and M.chelonae) or a combination thereof.

In another aspect, a method for treating or providing prophylaxisagainst a pulmonary infection in a patient is provided. In oneembodiment, the method comprises aerosolizing a pharmaceuticalformulation comprising a liposomal complexed aminoglycoside, wherein thepharmaceutical formulation is an aqueous dispersion of liposomes (e.g.,a liposomal solution or liposomal suspension), and is aerosolized at arate greater than about 0.53 gram per minute. The method furthercomprises administering the aerosolized pharmaceutical formulation tothe lungs of the patient; wherein the aerosolized pharmaceuticalformulation comprises a mixture of free aminoglycoside and liposomalcomplexed aminoglycoside, and the lipid component of the liposomeconsists of electrically neutral lipids. In a further embodiment, themass median aerodynamic diameter (MMAD) of the aerosol is about 1.0 μmto about 4.2 μm as measured by the ACI. In any one of the proceedingembodiments, the MMAD of the aerosol is about 3.2 μm to about 4.2 μm asmeasured by the ACI. In any one of the proceeding embodiments, the MMADof the aerosol is about 1.0 μm to about 4.9 μm as measured by the NGI.In any one of the proceeding embodiments, the MMAD of the aerosol isabout 4.4 μm to about 4.9 μm as measured by the NGI.

In one embodiment, the method comprises aerosolizing a pharmaceuticalformulation comprising a liposomal complexed aminoglycoside, wherein thepharmaceutical formulation is an aqueous dispersion and is aerosolizedat a rate greater than about 0.53 gram per minute. The method furthercomprises administering the aerosolized pharmaceutical formulation tothe lungs of the patient; wherein the aerosolized pharmaceuticalformulation comprises a mixture of free aminoglycoside and liposomalcomplexed aminoglycoside (e.g., aminoglycoside encapsulated in aliposome), and the liposome component of the formulation consists ofelectrically neutral lipids. In even a further embodiment, fine particlefraction (FPF) of the aerosol is greater than or equal to about 64%, asmeasured by the ACI, or greater than or equal to about 51%, as measuredby the NGI.

In another aspect, a liposomal complexed aminoglycoside aerosol (e.g., aliposomal complexed aminoglycoside) is provided. In one embodiment, theaerosol comprises an aminoglycoside and a plurality of liposomescomprising DPPC and cholesterol, wherein about 65% to about 75% of theaminoglycoside is liposomal complexed and the aerosol is generated at arate greater than about 0.53 gram per minute. In a further embodiment,about 65% to about 75% of the aminoglycoside is liposomal complexed, andthe aerosol is generated at a rate greater than about 0.53 gram perminute. In any one of the proceeding embodiments, the aerosol isgenerated at a rate greater than about 0.54 gram per minute. In any oneof the proceeding embodiments, the aerosol is generated at a rategreater than about 0.55 gram per minute. In any one of the precedingembodiments, the aminoglycoside is selected from an aminoglycosideprovided in Table A.

In one embodiment, the MMAD of the liposomal complexed aminoglycosideaerosol is about 3.2 μm to about 4.2 μm, as measured by the ACI, orabout 4.4 μm to about 4.9 μm, as measured by the NGI. In a furtherembodiment, the aerosol comprises an aminoglycoside and a plurality ofliposomes comprising DPPC and cholesterol, wherein about 65% to about75% of the aminoglycoside is liposomal complexed (e.g., encapsulated inthe plurality of the liposomes), and the liposomal aminoglycosideaerosol is generated at a rate greater than about 0.53 gram per minute.In a further embodiment, the aminoglycoside is selected from anaminoglycoside provided in Table A.

In one embodiment, the FPF of the lipid-complexed aminoglycoside aerosolis greater than or equal to about 64%, as measured by the AndersonCascade Impactor (ACI), or greater than or equal to about 51%, asmeasured by the Next Generation Impactor (NGI). In a further embodiment,the aerosol comprises an aminoglycoside and a plurality of liposomescomprising DPPC and cholesterol, wherein about 65% to about 75% of theaminoglycoside is liposomal complexed, for example, encapsulated in theplurality of the liposomes, and the liposomal aminoglycoside aerosol isgenerated at a rate greater than about 0.53 gram per minute. In any oneof the proceeding embodiments, the aerosol is generated at a rategreater than about 0.54 gram per minute. In any one of the proceedingembodiments, the aerosol is generated at a rate or greater than about0.55 gram per minute. In any of the preceding embodiments, theaminoglycoside is selected from an aminoglycoside provided in Table A.

In one embodiment, the aerosol comprises an aminoglycoside and aplurality of liposomes comprising DPPC and cholesterol, wherein about65% to about 75% of the aminoglycoside is liposomal complexed. In afurther embodiment, about 65% to about 75% of the aminoglycoside isencapsulated in the plurality of liposomes. In a further embodiment, theaerosol is generated at a rate greater than about 0.53 gram per minute,greater than about 0.54 gram per minute, or greater than about 0.55 gramper minute. In a further embodiment, the aminoglycoside is amikacin(e.g., amikacin sulfate).

In one embodiment, the concentration of the aminoglycoside in theliposomal complexed aminoglycoside is about 50 mg/mL or greater. In afurther embodiment, the concentration of the aminoglycoside in theliposomal complexed aminoglycoside is about 60 mg/mL or greater. In afurther embodiment, the concentration of the aminoglycoside in theliposomal complexed aminoglycoside is about 70 mg/mL or greater, forexample about 70 mg/mL to about 75 mg/mL. In a further embodiment, theaminoglycoside is selected from an aminoglycoside provided in Table A.In even a further embodiment, the aminoglycoside is amikacin (e.g.,amikacin sulfate).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a diagram of a nebulizer (aerosol generator) in which thepresent invention may be implemented.

FIG. 2 is an enlarged representation of the nebulizer diagram shown inFIG. 1.

FIG. 3 shows a cross-sectional view of a generally known aerosolgenerator, as described in WO 2001/032246.

FIG. 4 is an image of a PARI eFlow® nebulizer, modified for use with theaminoglycoside formulations described herein, and a blown up diagram ofthe nebulizer's membrane.

FIG. 5 is a cross-sectional computed tomography (CT) image showing amembrane having a relatively long nozzle portion.

FIG. 6 is a cross-sectional computed tomography (CT) image of astainless steel membrane having a relatively short nozzle portion.

FIG. 7 is a cross sectional cartoon depiction of the sputum/biofilmseen, for example, in patients with cystic fibrosis.

FIG. 8 is a graph of the time period of aerosol generation upon completeemission of the liquid within the liquid reservoir (Nebulization time)as a function of the initial gas cushion within the liquid reservoir(V_(A)).

FIG. 9 is a graph of negative pressure in the nebulizer as a function ofthe time of aerosol generation until complete emission of thepharmaceutical formulation from the liquid reservoir (nebulizationtime).

FIG. 10 is a graph of aerosol generation efficiency as a function of thenegative pressure in the nebulizer.

FIG. 11 is a graph of the period of time for aerosol generation uponcomplete emission of the liquid (nebulization time) as a function of theratio between the increased volume V_(RN) of the liquid reservoir andthe initial volume of liquid within the liquid reservoir (V_(L))(V_(RN)/V_(L)).

FIG. 12 is a graph showing the MMAD of aerosolized formulations as afunction of nebulization rate of the respective formulation.

FIG. 13 is a graph showing the FPF of aerosolized formulations as afunction of the nebulization rate of the respective formulation.

FIG. 14 is a schematic of the system used for the recovery of aerosolfor post-nebulization studies.

DETAILED DESCRIPTION OF THE INVENTION

The invention described herein is directed, in part, to systems foradministering an aminoglycoside pharmaceutical formulation to the lungsof a subject, for example, to treat a pulmonary disorder.

The term “treating” includes: (1) preventing or delaying the appearanceof clinical symptoms of the state, disorder or condition developing inthe subject that may be afflicted with or predisposed to the state,disorder or condition but does not yet experience or display clinical orsubclinical symptoms of the state, disorder or condition; (2) inhibitingthe state, disorder or condition (i.e., arresting, reducing or delayingthe development of the disease, or a relapse thereof in case ofmaintenance treatment, of at least one clinical or subclinical symptomthereof); and/or (3) relieving the condition (i.e., causing regressionof the state, disorder or condition or at least one of its clinical orsubclinical symptoms). The benefit to a subject to be treated is eitherstatistically significant or at least perceptible to the subject or tothe physician.

In one embodiment, pulmonary infections caused by the following bacteriaare treatable with the systems and formulations provided herein:Pseudomonas (e.g., P. aeruginosa, P. paucimobilis, P. putida, P.fluorescens, and P. acidovorans), Burkholderia (e.g., B. pseudomallei,B. cepacia, B. cepacia complex, B. dolosa, B. fungorum, B. gladioli, B.multivorans, B. vietnamiensis, B. pseudomallei, B. ambifaria, B.andropogonis, B. anthina, B. brasilensis, B. caledonica, B. caribensis,B. caryophylli), Staphylococcus (e.g., S. aureus, S. auricularis, S.carnosus, S. epidermidis, S. lugdunensis), Methicillin-resistantStaphylococcus aureus (MRSA), Streptococcus (e.g., Streptococcuspneumoniae), Escherichia coli, Klebsiella, Enterobacter, Serratia,Haemophilus, Yersinia pestis, Mycobacterium (e.g., nontuberculousmycobacterium).

In one embodiment, a patient is treated for a nontuberculousmycobacterial lung infection with one of the systems provided herein. Ina further embodiment, the nontuberculous mycobacterial lung infection isa recalcitrant nontuberculous mycobacterial lung infection.

In one embodiment, the systems provided herein are used to treat apatient having a pulmonary infection caused by Pseudomonas. In a furtherembodiment, the pulmonary infection is caused by a Pseudomonas speciesselected from a species provided in Table B, below.

TABLE B P. abietaniphila P. aeruginosa P. agarici P. alcaligenes P.alcaliphila P. amygdale P. anguilliseptica P. antarctica P.argentinensis P. asplenii P. aurantiaca P. aureofaciens P. avellanae P.azotifigens P. azotoformans P. balearica P. borbori P. brassicacearum P.brenneri P. cannabina P. caricapapayae P. cedrina P. chlororaphis P.cichorii P. citronellolis P. coenobios P. congelans P. coronofaciens P.corrugate P. costantinii P. cremoricolorata P. cruciviae P. delhiensisP. denitrificans P. excibis P. extremorientalis P. ficuseructae P.flavescens P. fluorescens P. fragi P. frederiksbergensis P. fulva P.fuscovaginae P. gelidicola P. gessardii P. grimontii P. indica P.jessenii P. jinjuensis P. kilonensis P. knackmussii P. koreensis P.libanensis P. lini P. lundensis P. lutea P. luteola P. mandelii P.marginalis P. mediterranea P. meliae P. mendocina P. meridiana P.migulae P. monteilii P. moraviensis P. mosselii P. mucidolens P.nitroreducens P. oleovorans P. orientalis P. oryzihabitans P. otitidisP. pachastrellae P. palleroniana P. panacis P. papaveris P. parafulva P.peli P. perolens P. pertucinogena P. plecoglossicida P. poae P.pohangensis P. proteolytica P. pseudoalcaligenes P. psychrophila P.psychrotolerans P. putida P. rathonis P. reptilivora P. resiniphila P.resinovorans P. rhizosphaerae P. rhodesiae P. rubescens P. salomonii P.savastanoi P. segitis P. septic P. simiae P. straminea P. stutzeri P.suis P. synxantha P. syringae P. taetrolens P. thermotolerans P.thivervalensis P. tolaasii P. tremae P. trivialis P. turbinellae P.tuticorinensis P. umsongensis P. vancouverensis P. veronii P.viridiflava P. vranovensis P. xanthomarina

The nontuberculous mycobacterial lung infection, in one embodiment, isselected from M. avium, M. avium subsp. hominissuis (MAH), M. abscessus,M. chelonae, M. bolletii, M. kansasii, M. ulcerans, M. avium, M. aviumcomplex (MAC) (M. avium and M. intracellulare), M. conspicuum, M.kansasii, M. peregrinum, M. immunogenum, M. xenopi, M. marinum, M.malmoense, M. marinum, M. mucogenicum, M. nonchromogenicum, M.scrofulaceum, M. simiae, M. smegmatis, M. szulgai, M. terrae, M. terraecomplex, M. haemophilum, M. genavense, M. asiaticum, M. shimoidei, M.gordonae, M. nonchromogenicum, M. triplex, M. lentiflavum, M. celatum,M. fortuitum, M. fortuitum complex (M. fortuitum and M. chelonae) or acombination thereof. In a further embodiment, the nontuberculousmycobacterial lung infection is M. abscessus or M. avium. In a furtherembodiment, the M. avium infection is M. avium subsp. hominissuis. Inone embodiment, the nontuberculous mycobacterial lung infection is arecalcitrant nontuberculous mycobacterial lung infection.

In another embodiment, a cystic fibrosis patient is treated for abacterial infection with one of the systems provided herein. In afurther embodiment, the bacterial infection is a lung infection due toPseudomonas aeruginosa. In yet another embodiment, a patient is treatedfor a pulmonary infection associated with bronchiectasis with one of thesystems provided herein.

“Prophylaxis,” as used herein, can mean complete prevention of aninfection or disease, or prevention of the development of symptoms ofthat infection or disease; a delay in the onset of an infection ordisease or its symptoms; or a decrease in the severity of a subsequentlydeveloped infection or disease or its symptoms.

The term “antibacterial” is art-recognized and refers to the ability ofthe compounds of the present invention to prevent, inhibit or destroythe growth of microbes of bacteria. Examples of bacteria are providedabove.

The term “antimicrobial” is art-recognized and refers to the ability ofthe aminoglycoside compounds of the present invention to prevent,inhibit, delay or destroy the growth of microbes such as bacteria,fungi, protozoa and viruses.

“Effective amount” means an amount of an aminoglycoside (e.g., amikacin)used in the present invention sufficient to result in the desiredtherapeutic response. The effective amount of the formulation providedherein comprises both free and liposomal complexed aminoglycoside. Forexample, the liposomal complexed aminoglycoside, in one embodiment,comprises aminoglycoside encapsulated in a liposome, or complexed with aliposome, or a combination thereof.

In one embodiment, the aminoglycoside is selected from amikacin,apramycin, arbekacin, astromicin, capreomycin, dibekacin, framycetin,gentamicin, hygromycin B, isepamicin, kanamycin, neomycin, netilmicin,paromomycin, rhodestreptomycin, ribostamycin, sisomicin, spectinomycin,streptomycin, tobramycin or verdamicin. In another embodiment, theaminoglycoside is selected from an aminoglycoside set forth in Table C,below.

TABLE C AC4437 amikacin arbekacin apramycin astromicin bekanamycinboholmycin brulamycin capreomycin dibekacin dactimicin etimicinframycetin gentamicin H107 hygromycin hygromycin B inosamycin K-4619isepamicin KA-5685 kanamycin neomycin netilmicin paromomycin plazomicinribostamycin sisomicin rhodestreptomycin sorbistin spectinomycinsporaricin streptomycin tobramycin verdamicin vertilmicin

In one embodiment, the aminoglycoside is an aminoglycoside free base, orits salt, solvate, or other non-covalent derivative. In a furtherembodiment, the aminoglycoside is amikacin. Included as suitableaminoglycosides used in the drug formulations of the present inventionare pharmaceutically acceptable addition salts and complexes of drugs.In cases where the compounds may have one or more chiral centers, unlessspecified, the present invention comprises each unique racemic compound,as well as each unique nonracemic compound. In cases in which the activeagents have unsaturated carbon-carbon double bonds, both the cis (Z) andtrans (E) isomers are within the scope of this invention. In cases wherethe active agents exist in tautomeric forms, such as keto-enoltautomers, each tautomeric form is contemplated as being included withinthe invention. Amikacin, in one embodiment, is present in thepharmaceutical formulation as amikacin base, or amikacin salt, forexample, amikacin sulfate or amikacin disulfate. In one embodiment, acombination of one or more of the above aminoglycosides is used in theformulations, systems and methods described herein. In a furtherembodiment, the combination comprises amikacin.

The therapeutic response can be any response that a user (e.g., aclinician) will recognize as an effective response to the therapy. Thetherapeutic response will generally be a reduction, inhibition, delay orprevention in growth of or reproduction of one or more bacterium, or thekilling of one or more bacterium, as described above. A therapeuticresponse may also be reflected in an improvement in pulmonary function,for example forced expiratory volume in one second (FEV₁). It is furtherwithin the skill of one of ordinary skill in the art to determineappropriate treatment duration, appropriate doses, and any potentialcombination treatments, based upon an evaluation of therapeuticresponse.

“Liposomal dispersion” refers to a solution or suspension comprising aplurality of liposomes.

An “aerosol”, as used herein, is a gaseous suspension of liquidparticles. The aerosol provided herein comprises particles of theliposomal dispersion.

A “nebulizer” or an “aerosol generator” is a device that converts aliquid into an aerosol of a size that can be inhaled into therespiratory tract. Pneumonic, ultrasonic, electronic nebulizers, e.g.,passive electronic mesh nebulizers, active electronic mesh nebulizersand vibrating mesh nebulizers are amenable for use with the invention ifthe particular nebulizer emits an aerosol with the required properties,and at the required output rate.

The process of pneumatically converting a bulk liquid into smalldroplets is called atomization. The operation of a pneumatic nebulizerrequires a pressurized gas supply as the driving force for liquidatomization. Ultrasonic nebulizers use electricity introduced by apiezoelectric element in the liquid reservoir to convert a liquid intorespirable droplets. Various types of nebulizers are described inRespiratory Care, Vol. 45, No. 6, pp. 609-622 (2000), the disclosure ofwhich is incorporated herein by reference in its entirety. The terms“nebulizer” and “aerosol generator” are used interchangeably throughoutthe specification. “Inhalation device”, “inhalation system” and“atomizer” are also used in the literature interchangeably with theterms “nebulizer” and “aerosol generator”.

“Fine particle fraction” or “FPF”, as used herein, refers to thefraction of the aerosol having a particle size less than 5 μm indiameter, as measured by cascade impaction. FPF is usually expressed asa percentage.

“Mass median diameter” or “MMD” is determined by laser diffraction orimpactor measurements, and is the average particle diameter by mass.

“Mass median aerodynamic diameter” or “MMAD” is normalized regarding theaerodynamic separation of aqua aerosol droplets and is determinedimpactor measurements, e.g., the Anderson Cascade Impactor (ACI) or theNext Generation Impactor (NGI). The gas flow rate, in one embodiment, is28 Liter per minute by the Anderson Cascade Impactor (ACI) and 15 Literper minute by the Next Generation Impactor (NGI). “Geometric standarddeviation” or “GSD” is a measure of the spread of an aerodynamicparticle size distribution.

In one embodiment, the present invention provides a system for treatinga pulmonary infection or providing prophylaxis against a pulmonaryinfection. Treatment is achieved via delivery of the aminoglycosideformulation by inhalation via nebulization. In one embodiment, thepharmaceutical formulation comprises an aminoglycoside agent, e.g., anaminoglycoside.

The pharmaceutical formulation, as provided herein, is a liposomaldispersion. Specifically, the pharmaceutical formulation is a dispersioncomprising a “liposomal complexed aminoglycoside” or an “aminoglycosideencapsulated in a liposome”. A “liposomal complexed aminoglycoside”includes embodiments where the aminoglycoside (or combination ofaminoglycosides) is encapsulated in a liposome, and includes any form ofaminoglycoside composition where at least about 1% by weight of theaminoglycoside is associated with the liposome either as part of acomplex with a liposome, or as a liposome where the aminoglycoside maybe in the aqueous phase or the hydrophobic bilayer phase or at theinterfacial headgroup region of the liposomal bilayer.

In one embodiment, the lipid component of the liposome compriseselectrically neutral lipids, positively charged lipids, negativelycharged lipids, or a combination thereof. In another embodiment, thelipid component comprises electrically neutral lipids. In a furtherembodiment, the lipid component consists essentially of electricallyneutral lipids. In even a further embodiment, the lipid componentconsists of electrically neutral lipids, e.g., a sterol and aphospholipid.

As provided above, liposomal complexed aminoglycoside embodimentsinclude embodiments where the aminoglycoside is encapsulated in aliposome. In addition, the liposomal complexed aminoglycoside describesany composition, solution or suspension where at least about 1% byweight of the aminoglycoside is associated with the lipid either as partof a complex with the liposome, or as a liposome where theaminoglycoside may be in the aqueous phase or the hydrophobic bilayerphase or at the interfacial headgroup region of the liposomal bilayer.In one embodiment, prior to nebulization, at least about 5%, at leastabout 10%, at least about 20%, at least about 25%, at least about 50%,at least about 75%, at least about 80%, at least about 85%, at leastabout 90% or at least about 95% of the aminoglycoside in the formulationis so associated. Association, in one embodiment, is measured byseparation through a filter where lipid and lipid-associated drug isretained (i.e., in the retentate) and free drug is in the filtrate.

The formulations, systems and methods provided herein comprise alipid-encapsulated or lipid-associated aminoglycoside agent. The lipidsused in the pharmaceutical formulations of the present invention can besynthetic, semi-synthetic or naturally-occurring lipids, includingphospholipids, tocopherols, sterols, fatty acids, negatively-chargedlipids and cationic lipids.

In one embodiment, at least one phospholipid is present in thepharmaceutical formulation. In one embodiment, the phospholipid isselected from: phosphatidylcholine (EPC), phosphatidylglycerol (PG),phosphatidylinositol (PI), phosphatidylserine (PS),phosphatidylethanolamine (PE), and phosphatidic acid (PA); the soyacounterparts, soy phosphatidylcholine (SPC); SPG, SPS, SPI, SPE, andSPA; the hydrogenated egg and soya counterparts (e.g., HEPC, HSPC),phospholipids made up of ester linkages of fatty acids in the 2 and 3 ofglycerol positions containing chains of 12 to 26 carbon atoms anddifferent head groups in the 1 position of glycerol that includecholine, glycerol, inositol, serine, ethanolamine, as well as thecorresponding phosphatidic acids. The carbon chains on these fatty acidscan be saturated or unsaturated, and the phospholipid may be made up offatty acids of different chain lengths and different degrees ofunsaturation.

In one embodiment, the pharmaceutical formulation includesdipalmitoylphosphatidylcholine (DPPC), a major constituent ofnaturally-occurring lung surfactant. In one embodiment, the lipidcomponent of the pharmaceutical formulation comprises DPPC andcholesterol, or consists essentially of DPPC and cholesterol, orconsists of DPPC and cholesterol. In a further embodiment, the DPPC andcholesterol have a mole ratio in the range of from about 19:1 to about1:1, or about 9:1 to about 1:1, or about 4:1 to about 1:1, or about 2:1to about 1:1, or about 1.86:1 to about 1:1. In even a furtherembodiment, the DPPC and cholesterol have a mole ratio of about 2:1 orabout 1:1. In one embodiment, DPPC and cholesterol are provided in anaminoglycoside formulation, e.g., an aminoglycoside formulation.

Other examples of lipids for use with the invention include, but are notlimited to, dimyristoylphosphatidycholine (DMPC),dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidcholine(DPPC), dipalmitoylphosphatidylglycerol (DPPG),distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol(DSPG), dioleylphosphatidyl-ethanolamine (DOPE), mixed phospholipidssuch as palmitoylstearoylphosphatidyl-choline (PSPC), and singleacylated phospholipids, for example,mono-oleoyl-phosphatidylethanolamine (MOPE).

In one embodiment, the at least one lipid component comprises a sterol.In a further embodiment, the at least one lipid component comprises asterol and a phospholipid, or consists essentially of a sterol and aphospholipid, or consists of a sterol and a phospholipid. Sterols foruse with the invention include, but are not limited to, cholesterol,esters of cholesterol including cholesterol hemi-succinate, salts ofcholesterol including cholesterol hydrogen sulfate and cholesterolsulfate, ergosterol, esters of ergosterol including ergosterolhemi-succinate, salts of ergosterol including ergosterol hydrogensulfate and ergosterol sulfate, lanosterol, esters of lanosterolincluding lanosterol hemi-succinate, salts of lanosterol includinglanosterol hydrogen sulfate, lanosterol sulfate and tocopherols. Thetocopherols can include tocopherols, esters of tocopherols includingtocopherol hemi-succinates, salts of tocopherols including tocopherolhydrogen sulfates and tocopherol sulfates. The term “sterol compound”includes sterols, tocopherols and the like.

In one embodiment, at least one cationic lipid (positively chargedlipid) is provided in the systems described herein. The cationic lipidsused can include ammonium salts of fatty acids, phospholids andglycerides. The fatty acids include fatty acids of carbon chain lengthsof 12 to 26 carbon atoms that are either saturated or unsaturated. Somespecific examples include: myristylamine, palmitylamine, laurylamine andstearylamine, dilauroyl ethylphosphocholine (DLEP), dimyristoylethylphosphocholine (DMEP), dipalmitoyl ethylphosphocholine (DPEP) anddistearoyl ethylphosphocholine (DSEP),N-(2,3-di-(9-(Z)-octadecenyloxy)-prop-1-yl-N,N,N-trimethylammoniumchloride (DOTMA) and 1,2-bis(oleoyloxy)-3-(trimethylammonio) propane(DOTAP).

In one embodiment, at least one anionic lipid (negatively charged lipid)is provided in the systems described herein. The negatively-chargedlipids which can be used include phosphatidyl-glycerols (PGs),phosphatidic acids (PAs), phosphatidylinositols (PIs) and thephosphatidyl serines (PSs). Examples include DMPG, DPPG, DSPG, DMPA,DPPA, DSPA, DMPI, DPPI, DSPI, DMPS, DPPS and DSPS.

Without wishing to be bound by theory, phosphatidylcholines, such asDPPC, aid in the uptake of the aminoglycoside agent by the cells in thelung (e.g., the alveolar macrophages) and helps to maintain theaminoglycoside agent in the lung. The negatively charged lipids such asthe PGs, PAs, PSs and PIs, in addition to reducing particle aggregation,are thought to play a role in the sustained activity characteristics ofthe inhalation formulation as well as in the transport of theformulation across the lung (transcytosis) for systemic uptake. Thesterol compounds, without wishing to be bound by theory, are thought toaffect the release characteristics of the formulation.

Liposomes are completely closed lipid bilayer membranes containing anentrapped aqueous volume. Liposomes may be unilamellar vesicles(possessing a single membrane bilayer) or multilamellar vesicles(onion-like structures characterized by multiple membrane bilayers, eachseparated from the next by an aqueous layer) or a combination thereof.The bilayer is composed of two lipid monolayers having a hydrophobic“tail” region and a hydrophilic “head” region. The structure of themembrane bilayer is such that the hydrophobic (nonpolar) “tails” of thelipid monolayers orient toward the center of the bilayer while thehydrophilic “heads” orient towards the aqueous phase.

Liposomes can be produced by a variety of methods (see, e.g., Cullis etal. (1987)). In one embodiment, one or more of the methods described inU.S. Patent Application Publication No. 2008/0089927 are used herein toproduce the aminoglycoside encapsulated lipid formulations (liposomaldispersion). The disclosure of U.S. Patent Application Publication No.2008/0089927 is incorporated by reference in its entirety for allpurposes. For example, in one embodiment, at least one lipid and anaminoglycoside are mixed with a coacervate (i.e., a separate liquidphase) to form the liposome formulation. The coacervate can be formed toprior to mixing with the lipid, during mixing with the lipid or aftermixing with the lipid. Additionally, the coacervate can be a coacervateof the active agent.

In one embodiment, the liposomal dispersion is formed by dissolving oneor more lipids in an organic solvent forming a lipid solution, and theaminoglycoside coacervate forms from mixing an aqueous solution of theaminoglycoside with the lipid solution. In a further embodiment, theorganic solvent is ethanol. In even a further embodiment, the one ormore lipids comprise a phospholipid and a sterol.

In one embodiment, liposomes are produces by sonication, extrusion,homogenization, swelling, electroformation, inverted emulsion or areverse evaporation method. Bangham's procedure (J. Mol. Biol. (1965))produces ordinary multilamellar vesicles (MLVs). Lenk et al. (U.S. Pat.Nos. 4,522,803, 5,030,453 and 5,169,637), Fountain et al. (U.S. Pat. No.4,588,578) and Cullis et al. (U.S. Pat. No. 4,975,282) disclose methodsfor producing multilamellar liposomes having substantially equalinterlamellar solute distribution in each of their aqueous compartments.Paphadjopoulos et al., U.S. Pat. No. 4,235,871, discloses preparation ofoligolamellar liposomes by reverse phase evaporation. Each of themethods is amenable for use with the present invention.

Unilamellar vesicles can be produced from MLVs by a number oftechniques, for example, the extrusion techniques of U.S. Pat. Nos.5,008,050 and 5,059,421. Sonication and homogenization cab be so used toproduce smaller unilamellar liposomes from larger liposomes (see, forexample, Paphadjopoulos et al. (1968); Deamer and Uster (1983); andChapman et al. (1968)).

The liposome preparation of Bangham et al. (J. Mol. Biol. 13, 1965, pp.238-252) involves suspending phospholipids in an organic solvent whichis then evaporated to dryness leaving a phospholipid film on thereaction vessel. Next, an appropriate amount of aqueous phase is added,the 60 mixture is allowed to “swell”, and the resulting liposomes whichconsist of multilamellar vesicles (MLVs) are dispersed by mechanicalmeans. This preparation provides the basis for the development of thesmall sonicated unilamellar vesicles described by Papahadjopoulos et al.(Biochim. Biophys. Acta. 135, 1967, pp. 624-638), and large unilamellarvesicles.

Techniques for producing large unilamellar vesicles (LUVs), such as,reverse phase evaporation, infusion procedures, and detergent dilution,can be used to produce liposomes for use in the pharmaceuticalformulations provided herein. A review of these and other methods forproducing liposomes may be found in the text Liposomes, Marc Ostro, ed.,Marcel Dekker, Inc., New York, 1983, Chapter 1, which is incorporatedherein by reference. See also Szoka, Jr. et al., (Ann. Rev. Biophys.Bioeng. 9, 1980, p. 467), which is also incorporated herein by referencein its entirety for all purposes.

Other techniques for making liposomes include those that formreverse-phase evaporation vesicles (REV), U.S. Pat. No. 4,235,871.Another class of liposomes that may be used is characterized as havingsubstantially equal lamellar solute distribution. This class ofliposomes is denominated as stable plurilamellar vesicles (SPLV) asdefined in U.S. Pat. No. 4,522,803, and includes monophasic vesicles asdescribed in U.S. Pat. No. 4,588,578, and frozen and thawedmultilamellar vesicles (FATMLV) as described above.

A variety of sterols and their water soluble derivatives such ascholesterol hemisuccinate have been used to form liposomes; see, e.g.,U.S. Pat. No. 4,721,612. Mayhew et al., PCT Publication No. WO 85/00968,described a method for reducing the toxicity of drugs by encapsulatingthem in liposomes comprising alpha-tocopherol and certain derivativesthereof. Also, a variety of tocopherols and their water solublederivatives have been used to form liposomes, see PCT Publication No.87/02219.

The pharmaceutical formulation, in one embodiment, pre-nebulization,comprises liposomes with a mean diameter, that is measured by a lightscattering method, of approximately 0.01 microns to approximately 3.0microns, for example, in the range about 0.2 to about 1.0 microns. Inone embodiment, the mean diameter of the liposomes in the formulation isabout 200 nm to about 300 nm, about 210 nm to about 290 nm, about 220 nmto about 280 nm, about 230 nm to about 280 nm, about 240 nm to about 280nm, about 250 nm to about 280 nm or about 260 nm to about 280 nm. Thesustained activity profile of the liposomal product can be regulated bythe nature of the lipid membrane and by inclusion of other excipients inthe composition.

In order to minimize dose volume and reduce patient dosing time, in oneembodiment, it is important that liposomal entrapment of theaminoglycoside (e.g., the aminoglycoside amikacin) be highly efficientand that the L/D ratio be at as low a value as possible and/or practicalwhile keeping the liposomes small enough to penetrate patient mucus andbiofilms, e.g., Pseudomonas biofilms. In one embodiment, the L/D ratioin liposomes provided herein is 0.7 or about 0.7 (w/w). In a furtherembodiment, the liposomes provided herein are small enough toeffectively penetrate a bacterial biofilm (e.g., Pseudomonas biofilm).In even a further embodiment, the mean diameter of the liposomes, asmeasured by light scattering is about 260 to about 280 nm.

The lipid to drug ratio in the pharmaceutical formulations providedherein, in one embodiment, is 3 to 1 or less, 2.5 to 1 or less, 2 to 1or less, 1.5 to 1 or less, or 1 to 1 or less. The lipid to drug ratio inthe pharmaceutical formulations provided herein, in another embodiment,is less than 3 to 1, less than 2.5 to 1, less than 2 to 1, less than 1.5to 1, or less than 1 to 1. Ina further embodiment, the lipid to drugratio is about 0.7 to or less or about 0.7 to 1. In one embodiment, oneof the lipids or lipid combinations in Table 1, below, is used in thepharmaceutical formulation of the invention.

TABLE 1 Lipids amenable for use with the invention MoleLipid/aminoglycoside Lipid(s) ratio (w/w) DPPC — 1.1 DPPC/DOPG 9:1 1.0DPPC/DOPG 7:1 3.9 DPPC/DOPG 1:1 2.8 DPPC/DOPG 0.5:1   2.7 DOPG — 2.6DPPC/Cholesterol about 1:1 about 0.7 DPPC/Cholesterol 1:1 0.7DPPC/Cholesterol 19:1  1.0 DPPC/Cholesterol 9:1 1.2 DPPC/Cholesterol 4:11.7 DPPC/Cholesterol 1.86:1   2.1 DPPC/Cholesterol 1:1 2.7DPPC/DOPC/Cholesterol 8.55:1:0.45 2.0 DPPC/DOPC/Cholesterol 6.65:1:0.353.0 DPPC/DOPC/Cholesterol 19:20:1 2.5 DPPC/DOPC/Cholesterol 8.55:1:0.453.8 DPPC/DOPC/Cholesterol 6.65:1:0.35 4.1 DPPC/DOPC/Cholesterol 19:20:14.2 DPPC/DOPC/DOPG/Cholesterol 42:4:9:45 3.7 DPPC/DOPC/DOPG/Cholesterol59:5:6:30 3.7

In one embodiment, the system provided herein comprises anaminoglycoside formulation, for example, an amikacin formulation, e.g.,amikacin base formulation. In one embodiment, the amount ofaminoglycoside provided in the system is about 450 mg, about 500 mg,about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg,about 600 mg or about 610 mg. In another embodiment, the amount ofaminoglycoside provided in the system is from about 500 mg to about 600mg, or from about 500 mg to about 650 mg, or from about 525 mg to about625 mg, or from about 550 mg to about 600 mg. In one embodiment, theamount of aminoglycoside administered to the subject is about 560 mg andis provided in an 8 mL formulation. In one embodiment, the amount ofaminoglycoside administered to the subject is about 590 mg and isprovided in an 8 mL formulation. In one embodiment, the amount ofaminoglycoside administered to the subject is about 600 mg and isprovided in an 8 mL formulation. In one embodiment, the aminoglycosideis amikacin and the amount of amikacin provided in the system is about450 mg, about 500 mg, about 550 mg, about 560 mg, about 570 mg, about580 mg, about 590 mg, about 600 mg or about 610 mg. In anotherembodiment, the aminoglycoside is amikacin and the amount of amikacinprovided in the system is from about 500 mg to about 650 mg, or fromabout 525 mg to about 625 mg, or from about 550 mg to about 600 mg. Inone embodiment, the aminoglycoside is amikacin and the amount ofamikacin administered to the subject is about 560 mg, and is provided inan 8 mL formulation. In one embodiment, the aminoglycoside is amikacinand the amount of amikacin administered to the subject is about 590 mg,and is provided in an 8 mL formulation. In one embodiment, the theaminoglycoside is amikacin and the amount of aminoglycoside administeredto the subject is about 600 mg and is provided in an 8 mL formulation.

In one embodiment, the system provided herein comprises anaminoglycoside formulation, for example, an amikacin (base formulation).In one embodiment, the aminoglycoside formulation provided hereincomprises about 60 mg/mL aminoglycoside, about 65 mg/mL aminoglycoside,about 70 mg/mL aminoglycoside, about 75 mg/mL aminoglycoside, about 80mg/mL aminoglycoside, about 85 mg/mL aminoglycoside, or about 90 mg/mLaminoglycoside. In a further embodiment, the aminoglycoside is amikacin.

In one embodiment, the system provided herein comprises an about 8 mLliposomal amikacin formulation. In one embodiment, the density of theliposomal amikacin formulation is about 1.05 gram/mL; and in oneembodiment, approximately 8.4 grams of the liposomal amikacinformulation per dose is present in the system of the invention. In afurther embodiment, the entire volume of the formulation is administeredto a subject in need thereof.

In one embodiment, the pharmaceutical formulation provided hereincomprises at least one aminoglycoside, at least one phospholipid and asterol. In a further embodiment, the pharmaceutical formulationcomprises an aminoglycoside, DPPC and cholesterol. In one embodiment,the pharmaceutical formulation is the formulation provided in Table 2,below.

TABLE 2 Pharmaceutical Formulations Component Concentration ComponentConcentration Formulation A (pH 6.0-7.0) Formulation D (pH ~6.5)Aminoglycoside 60-80 mg/mL Aminoglycoside ~70 mg/mL Phospholipid 30-40mg/mL Phospholipid ~32-35 mg/mL Sterol 10-20 mg/mL Sterol ~16-17 mg/mLSalt 0.5%-5.0% Salt ~1.5% Formulation B (pH 6.0-7.0) Formulation E (pH~6.5) Amikacin 60-80 mg/mL Amikacin ~70 mg/mL DPPC 30-40 mg/mL DPPC~32-35 mg/mL Cholesterol 10-20 mg/mL Cholesterol ~16-17 mg/mL NaCl0.5%-5.0% NaCl ~1.5% Formulation C (pH 6.0-7.0) Formulation F (pH ~6.5)Amikacin 70-80 mg/mL Amikacin ~70 mg/mL DPPC 35-40 mg/mL DPPC ~30-35mg/mL Cholesterol 15-20 mg/mL Cholesterol ~15-17 mg/mL NaCl 0.5%-5.0%NaCl ~1.5%

It should be noted that increasing aminoglycoside concentration alonemay not result in a reduced dosing time. For example, in one embodiment,the lipid to drug ratio is fixed, and as amikacin concentration isincreased (and therefore lipid concentration is increased, since theratio of the two is fixed, for example at ˜0.7:1), the viscosity of thesolution also increases, which slows nebulization time.

In one embodiment, prior to nebulization of the aminoglycosideformulation, about 70% to about 100% of the aminoglycoside present inthe formulation is liposomal complexed. In a further embodiment, theaminoglycoside is an aminoglycoside. In even a further embodiment, theaminoglycoside is amikacin. In another embodiment, prior tonebulization, about 80% to about 99%, or about 85% to about 99%, orabout 90% to about 99% or about 95% to about 99% or about 96% to about99% of the aminoglycoside present in the formulation is liposomalcomplexed. In a further embodiment, the aminoglycoside is amikacin ortobramycin. In even a further embodiment, the aminoglycoside isamikacin. In another embodiment, prior to nebulization, about 98% of theaminoglycoside present in the formulation is liposomal complexed. In afurther embodiment, the aminoglycoside is amikacin or tobramycin. Ineven a further embodiment, the aminoglycoside is amikacin.

In one embodiment, upon nebulization, about 20% to about 50% of theliposomal complexed aminoglycoside agent is released, due to shearstress on the liposomes. In a further embodiment, the aminoglycosideagent is an amikacin. In another embodiment, upon nebulization, about25% to about 45%, or about 30% to about 40% of the liposomal complexedaminoglycoside agent is released, due to shear stress on the liposomes.In a further embodiment, the aminoglycoside agent is amikacin.

As provided herein, the present invention provides methods and systemsfor treatment of lung infections by inhalation of a liposomalaminoglycoside formulation via nebulization. The formulation, in oneembodiment, is administered via a nebulizer, which provides an aerosolmist of the formulation for delivery to the lungs of a subject.

In one embodiment, the nebulizer described herein generates an aerosol(i.e., achieves a total output rate) of the aminoglycosidepharmaceutical formulation at a rate greater than about 0.53 g perminute, greater than about 0.54 g per minute, greater than about 0.55 gper minute, greater than about 0.58 g per minute, greater than about0.60 g per minute, greater than about 0.65 g per minute or greater thanabout 0.70 g per minute. In another embodiment, the nebulizer describedherein generates an aerosol (i.e., achieves a total output rate) of theaminoglycoside pharmaceutical formulation at about 0.53 g per minute toabout 0.80 g per minute, at about 0.53 g per minute to about 0.70 g perminute, about 0.55 g per min to about 0.70 g per minute, about 0.53 gper minute to about 0.65 g per minute, or about 0.60 g per minute toabout 0.70 g per minute. In yet another embodiment, the nebulizerdescribed herein generates an aerosol (i.e., achieves a total outputrate) of the aminoglycoside pharmaceutical formulation at about 0.53 gper minute to about 0.75 g per minute, about 0.55 g per min to about0.75 g per minute, about 0.53 g per minute to about 0.65 g per minute,or about 0.60 g per minute to about 0.75 g per minute.

Upon nebulization, the liposomes in the pharmaceutical formulation leakdrug. In one embodiment, the amount of liposomal complexedaminoglycoside post-nebulization is about 45% to about 85%, or about 50%to about 80% or about 51% to about 77%. These percentages are alsoreferred to herein as “percent associated aminoglycosidepost-nebulization”. As provided herein, in one embodiment, the liposomescomprise an aminoglycoside, e.g., amikacin. In one embodiment, thepercent associated aminoglycoside post-nebulization is from about 60% toabout 70%. In a further embodiment, the aminoglycoside is amikacin. Inanother embodiment, the percent associated aminoglycosidepost-nebulization is about 67%, or about 65% to about 70%. In a furtherembodiment, the aminoglycoside is amikacin.

In one embodiment, the percent associated aminoglycosidepost-nebulization is measured by reclaiming the aerosol from the air bycondensation in a cold-trap, and the liquid is subsequently assayed forfree and encapsulated aminoglycoside (associated aminoglycoside).

In one embodiment, the MMAD of the aerosol of the pharmaceuticalformulation is less than 4.9 μm, less than 4.5 μm, less than 4.3 μm,less than 4.2 μm, less than 4.1 μm, less than 4.0 μm or less than 3.5μm, as measured by the ACI at a gas flow rate of about 28 L/minute, orby the Next Generation Impactor NGI at a gas flow rate of about 15L/minute.

In one embodiment, the MMAD of the aerosol of the pharmaceuticalformulation is about 1.0 μm to about 4.2 μm, about 3.2 μm to about 4.2μm, about 3.4 μm to about 4.0 μm, about 3.5 μm to about 4.0 μm or about3.5 μm to about 4.2 μm, as measured by the ACI. In one embodiment, theMMAD of the aerosol of the pharmaceutical formulation is about 2.0 μm toabout 4.9 μm, about 4.4 μm to about 4.9 μm, about 4.5 μm to about 4.9μm, or about 4.6 μm to about 4.9 μm, as measured by the NGI.

In another embodiment, the nebulizer described herein generates anaerosol of the aminoglycoside pharmaceutical formulation at a rategreater than about 0.53 g per minute, greater than about 0.55 g perminute, or greater than about 0.60 g per minute or about 0.60 g perminute to about 0.70 g per minute. In a further embodiment, the FPF ofthe aerosol is greater than or equal to about 64%, as measured by theACI, greater than or equal to about 70%, as measured by the ACI, greaterthan or equal to about 51%, as measured by the NGI, or greater than orequal to about 60%, as measured by the NGI.

In one embodiment, the system provided herein comprises a nebulizerselected from an electronic mesh nebulizer, pneumonic (jet) nebulizer,ultrasonic nebulizer, breath-enhanced nebulizer and breath-actuatednebulizer. In one embodiment, the nebulizer is portable.

The principle of operation of a pneumonic nebulizer is generally knownto those of ordinary skill in the art and is described, e.g., inRespiratory Care, Vol. 45, No. 6, pp. 609-622 (2000). Briefly, apressurized gas supply is used as the driving force for liquidatomization in a pneumatic nebulizer. Compressed gas is delivered, whichcauses a region of negative pressure. The solution to be aerosolized isthen delivered into the gas stream and is sheared into a liquid film.This film is unstable and breaks into droplets because of surfacetension forces. Smaller particles, i.e., particles with the MMAD and FPFproperties described above, can then be formed by placing a baffle inthe aerosol stream. In one pneumonic nebulizer embodiment, gas andsolution is mixed prior to leaving the exit port (nozzle) andinteracting with the baffle. In another embodiment, mixing does not takeplace until the liquid and gas leave the exit port (nozzle). In oneembodiment, the gas is air, O₂ and/or CO₂.

In one embodiment, droplet size and output rate can be tailored in apneumonic nebulizer. However, consideration should be paid to theformulation being nebulized, and whether the properties of theformulation (e.g., % associated aminoglycoside) are altered due to themodification of the nebulizer. For example, in one embodiment, the gasvelocity and/or pharmaceutical formulation velocity is modified toachieve the output rate and droplet sizes of the present invention.Additionally or alternatively, the flow rate of the gas and/or solutioncan be tailored to achieve the droplet size and output rate of theinvention. For example, an increase in gas velocity, in one embodiment,decreased droplet size. In one embodiment, the ratio of pharmaceuticalformulation flow to gas flow is tailored to achieve the droplet size andoutput rate of the invention. In one embodiment, an increase in theratio of liquid to gas flow increases particle size.

In one embodiment, a pneumonic nebulizer output rate is increased byincreasing the fill volume in the liquid reservoir. Without wishing tobe bound by theory, the increase in output rate may be due to areduction of dead volume in the nebulizer. Nebulization time, in oneembodiment, is reduced by increasing the flow to power the nebulizer.See, e.g., Clay et al. (1983). Lancet 2, pp. 592-594 and Hess et al.(1996). Chest 110, pp. 498-505.

In one embodiment, a reservoir bag is used to capture aerosol during thenebulization process, and the aerosol is subsequently provided to thesubject via inhalation. In another embodiment, the nebulizer providedherein includes a valved open-vent design. In this embodiment, when thepatient inhales through the nebulizer, nebulizer output is increased.During the expiratory phase, a one-way valve diverts patient flow awayfrom the nebulizer chamber.

In one embodiment, the nebulizer provided herein is a continuousnebulizer. In other words, refilling the nebulizer with thepharmaceutical formulation while administering a dose is not needed.Rather, the nebulizer has at least an 8 mL capacity or at least a 10 mLcapacity.

In one embodiment, a vibrating mesh nebulizer is used to deliver theaminoglycoside formulation of the invention to a patient in needthereof. In one embodiment, the nebulizer membrane vibrates at anultrasonic frequency of about 100 kHz to about 250 kHz, about 110 kHz toabout 200 kHz, about 110 kHz to about 200 kHz, about 110 kHz to about150 kHz. In one embodiment, the nebulizer membrane vibrates at afrequency of about 117 kHz upon the application of an electric current.

In one embodiment, the nebulizer provided herein does not use an aircompressor and therefore does not generate an air flow. In oneembodiment, aerosol is produced by the aerosol head which enters themixing chamber of the device. When the patient inhales, air enters themixing chamber via one-way inhalation valves in the back of the mixingchamber and carries the aerosol through the mouthpiece to the patient.On exhalation, the patient's breath flows through the one-way exhalationvalve on the mouthpiece of the device. In one embodiment, the nebulizercontinues to generate aerosol into the mixing chamber which is thendrawn in by the subject on the next breath—and this cycle continuesuntil the nebulizer medication reservoir is empty.

Although not limited thereto, the present invention, in one embodiment,is carried out with one of the aerosol generators (nebulizers) depictedin FIGS. 1, 2, 3 and 4. Additionally, the systems of the invention, inone embodiment, include a nebulizer described in European PatentApplications 11169080.6 and/or 10192385.2. These applications areincorporated by reference in their entireties.

FIG. 1 shows a therapeutic aerosol device 1 with a nebulizing chamber 2,a mouthpiece 3 and a membrane aerosol generator 4 with an oscillatingmembrane 5. The oscillating membrane may, for example, be brought tooscillation by annular piezo elements (not shown), examples of which aredescribed in WO 1997/29851.

When in use, the pharmaceutical formulation is located on one side ofthe oscillating membrane 5, see FIGS. 1, 2 and 4, and this liquid isthen transported through openings in the oscillating membrane 5 andemitted on the other side of the oscillating membrane 5, see bottom ofFIG. 1, FIG. 2, as an aerosol into the nebulizing chamber 2. The patientis able to breathe in the aerosol present in the nebulizing chamber 2 atthe mouthpiece 3.

The oscillating membrane 5 comprises a plurality of through holes.Droplets of the aminoglycoside formulation are generated when theaminoglycoside pharmaceutical formulation passes through the membrane.In one embodiment, the membrane is vibratable, a so called activeelectronic mesh nebulizer, for example the eFlow® nebulizer from PARTPharma, HL100 nebulizer from Health and Life, or the Aeroneb Go® fromAerogen (Novartis). In a further embodiment, the membrane vibrates at anultrasonic frequency of about 100 kHz to about 150 kHz, about 110 kHz toabout 140 kHz, or about 110 kHz to about 120 kHz. In a furtherembodiment, the membrane vibrates at a frequency of about 117 kHz uponthe application of an electric current. In a further embodiment, themembrane is fixed and the a further part of the fluid reservoir or fluidsupply is vibratable, a so called passive electronic mesh nebulizer, forexample the MicroAir Electronic Nebulizer Model U22 from Omron or theI-Neb I-neb AAD Inhalation System from Philips Respironics.

In one embodiment, the length of the nozzle portion of the through holesformed in the membrane (e.g., vibratable membrane) influences the totaloutput rate (TOR) of the aerosol generator. In particular, it has beenfound that the length of the nozzle portion is directly proportional tothe total output rate, wherein the shorter the nozzle portion, thehigher the TOR and vice versa.

In one embodiment, the nozzle portion is sufficiently short and small indiameter as compared to the upstream portion of the through hole. In afurther embodiment, the length of the portions upstream of the nozzleportion within the through hole does not have a significant influence onthe TOR.

In one embodiment, the length of the nozzle portion influences thegeometric standard deviation (GSD) of the droplet size distribution ofthe aminoglycoside pharmaceutical formulation. Low GSDs characterize anarrow droplet size distribution (homogeneously sized droplets), whichis advantageous for targeting aerosol to the respiratory system, forexample for the treatment of bacterial infections (e.g., Pseudomonas orMycobacteria) in cystic fibrosis patients, or the treatment ofnontuberculosis mycobacteria, bronchiectasis (e.g., the treatment ofcystic fibrosis or non-cystic fibrosis patients), Pseudomonas orMycobacteria in patients. That is, the longer the nozzle portion thelower the GSD. The average droplet size, in one embodiment is less than5 μm, and has a GSD in a range of 1.0 to 2.2, or about 1.0 to about 2.2,or 1.5 to 2.2, or about 1.5 to about 2.2.

In one embodiment, as provided above, the system provided hereincomprises a nebulizer which generates an aerosol of the aminoglycosidepharmaceutical formulation at a rate greater than about 0.53 g perminute, or greater than about 0.55 g per minute. In a furtherembodiment, the nebulizer comprises a vibratable membrane having a firstside for being in contact with the fluid and an opposite second side,from which the droplets emerge.

The membrane, e.g., a stainless steel membrane, may be vibrated by meansof a piezoelectric actuator or any other suitable means. The membranehas a plurality of through holes penetrating the membrane in anextension direction from the first side to the second side. The throughholes may be formed as previously mentioned by a laser source,electroforming or any other suitable process. When the membrane isvibrating, the aminoglycoside pharmaceutical formulation passes thethrough holes from the first side to the second side to generate theaerosol at the second side. Each of the through holes, in oneembodiment, comprises an entrance opening and an exit opening. In afurther embodiment, each of the through holes comprises a nozzle portionextending from the exit opening over a portion of the through holestowards the entrance opening. The nozzle portion is defined by thecontinuous portion of the through hole in the extension directioncomprising a smallest diameter of the through hole and bordered by alarger diameter of the through hole. In one embodiment, the largerdiameter of the through hole is defined as that diameter that is closestto 3 times, about 3 times, 2 times, about 2 times, 1.5 times, or about1.5 times, the smallest diameter.

The smallest diameter of the through hole, in one embodiment, is thediameter of the exit opening. In another embodiment, the smallestdiameter of the through hole is a diameter about 0.5×, about 0.6×, about0.7×, about 0.8× or about 0.9× the diameter of the exit opening.

In one embodiment, the nebulizer provided herein comprises through holesin which the ratio of the total length of at least one of the throughholes in the extension direction to the length of the respective nozzleportion of the through hole in the extension direction is at least 4, orat least about 4, or at least 4.5, or at least about 4.5, or at least 5,or at least about 5, or greater than about 5. In another embodiment, thenebulizer provided herein comprises through holes in which the ratio ofthe total length of the majority of through holes in the extensiondirection to the length of the respective nozzle portion of the throughholes in the extension direction is at least 4, or at least about 4, orat least 4.5, or at least about 4.5, or at least 5, or at least about 5,or greater than about 5.

The extension ratios set forth above provide, in one embodiment, anincreased total output rate, as compared to previously known nebulizers,and also provides a sufficient GSD. The ratio configurations, in oneembodiment, achieve shorter application periods, leading to greatercomfort for the patient and effectiveness of the aminoglycosidecompound. This is particularly advantageous if the aminoglycosidecompound in the formulation, due to its properties, is prepared at a lowconcentration, and therefore, a greater volume of the aminoglycosidepharmaceutical formulation must be administered in an acceptable time,e.g., one dosing session.

According to one embodiment, the nozzle portion terminates flush withthe second side. Therefore, the length of the nozzle portion, in oneembodiment, is defined as that portion starting from the second sidetowards the first side up to and bordered by the diameter that it isclosest to about triple, about twice, about 2.5×, or about 1.5× thesmallest diameter. The smallest diameter, in this embodiment, is thediameter of the exit opening.

In one embodiment, the smallest diameter (i.e., one border of the nozzleportion) is located at the end of the nozzle portion in the extensiondirection adjacent to the second side. In one embodiment, the largerdiameter of the through hole, located at the other border of the nozzleportion, is located upstream of the smallest diameter in the directionin which the fluid passes the plurality of through holes duringoperation.

According to one embodiment, the smallest diameter is smaller than about4.5 μm, smaller than about 4.0 μm, smaller than about 3.5 μm, or smallerthan about 3.0 μm.

In one embodiment, the total length of at least one through hole in theextension direction is at least about 50 μm, at least about 60 μm, atleast about 70 μm, or at least about 80 μm. In a further embodiment, thetotal length of at least one of the plurality of through holes is atleast about 90 μm. In one embodiment, the total length of a majority ofthe plurality of through holes in the extension direction is at leastabout 50 μm, at least about 60 μm, at least about 70 μm, or at leastabout 80 μm. In a further embodiment, the total length of a majority ofthe plurality of through holes is at least about 90 μm.

The length of the nozzle portion, in one embodiment, is less than about25 μm, less than about 20 μm or less than about 15 μm.

According to one embodiment, the through holes are laser-drilled throughholes formed in at least two stages, one stage forming the nozzleportion and the remaining stage(s) forming the remainder of the throughholes.

In another embodiment, the manufacturing methods used lead to a nozzleportion which is substantially cylindrical or conical with a toleranceof less than +100% of the smallest diameter, less than +75% of thesmallest diameter, less than +50% of the smallest diameter, less than+30% of the smallest diameter, less than +25% of the smallest diameter,or less than +15% of the smallest diameter.

Alternatively or additionally, the through holes are formed in anelectroforming process. In one embodiment, the through holes have afirst funnel-shaped portion at the first side and a second funnel-shapedportion at the second side with the nozzle portion in-between the firstand the second funnel-shaped portions and defined between the exitopening and the larger diameter. In this instance, the total length ofthe through holes may as well be defined by the distances from the firstside to the exit opening (smallest diameter) only.

In addition, the total output rate (TOR) may be further increased byincreasing the number of through holes provided in the membrane. In oneembodiment, an increase in number of through holes is achieved byincreasing the active perforated surface of the membrane and maintainingthe distance of the through holes relative to each other at the samelevel. In another embodiment, the number of through holes is increasedby reducing the distance of the through holes relative to each other andmaintaining the active area of the membrane. In addition, a combinationof the above strategies may be used.

In one embodiment, the total output rate of the nebulizer describedherein is increased by increasing the density of through holes in themembrane. In one embodiment, the average distance between through holesis about 70 μm, or about 60 μm, or about 50 μm.

In one embodiment, the membrane comprises between about 200 and about8,000 through holes, between about 1,000 and about 6,000 through holes,between about 2,000 and about 5,000 through holes or about 2,000 andabout 4,000 through holes. In one embodiment, the number of throughholes described above increases the TOR, and the TOR is increasedregardless of whether the nozzle parameters are implemented as describedabove. In one embodiment, the nebulizer provided herein comprises about3,000 through holes. In a further embodiment, the through holes arelocated in a hexagonal array, e.g., at about the center of the membrane(e.g., stainless steel membrane). In a further embodiment, the averagedistance between through holes is about 70 μm.

FIG. 3 shows an aerosol generator (nebulizer) as disclosed in WO2001/032246, which is hereby incorporated by reference in its entirety.The aerosol generator comprises a fluid reservoir 21 to contain thepharmaceutical formulation, to be emitted into the mixing chamber 3 inthe form of an aerosol and to be inhaled by means of the mouth piece 4through the opening 41.

The aerosol generator comprises a vibratable membrane 22 vibrated bymeans of a piezoelectric actuator 23. The vibratable membrane 22 has afirst side 24 facing the fluid container 21 and a second opposite side25 facing the mixing chamber 3. In use, the first side 24 of thevibratable membrane 22 is in contact with the fluid contained in thefluid container 21. A plurality of through holes 26 penetrating themembrane from the first side 24 to the second side 25 are provided inthe membrane 22. In use, the fluid passes from the fluid container 21through the through holes 26 from the first 24 to the second side 25when the membrane 22 is vibrated for generating the aerosol at thesecond side 25 and emitting it into the mixing chamber 3. This aerosolmay then be drawn by inhalation of a patient from the mixing chamber 3via the mouth piece 4 and its inhalation opening 41.

FIG. 5 shows a cross-sectional computed tomography scan showing three ofthe through holes 26 of such a vibratable membrane 22. The through holes26 of this particular embodiment are formed by laser drilling usingthree stages of different process parameters, respectively. In a firststage, the portion 30 is formed. In a second stage the portion 31 isformed and in a third stage the nozzle portion 32 is formed. In thisparticular embodiment, the length of the nozzle portion 32 is about 26μm, whereas the portion 31 has a length of about 51 μm. The firstportion 30 has a length of about 24.5 μm. As a result, the total lengthof each through hole is the sum of the length of the portion 30, theportion 31 and the nozzle portion 32, that is in this particularexample, about 101.5 μm. Thus, the ratio of the total length of eachthrough hole 26 in the extension direction E to the length of arespective one of the nozzle portions 32 in the extension direction E isapproximately 3.9.

In the embodiment in FIG. 6, the first portion 30 has a length of about27 μm, the portion 31 a length of about 55 μm and a nozzle portion alength of about 19 μm. As a result, the total length of the through hole26 is about 101 μm. Thus, the ratio of the total length of the throughhole 26 to the length of the corresponding nozzle portion 32 in thisembodiment is approximately 5.3.

Both the vibratable membranes in FIGS. 5 and 6 were manufactured with6,000 through holes 26. The below table (Table 3) indicates the massmedian diameter (MMD), as determined by laser diffraction, of theparticles emitted at the second side of the membrane, the time requiredfor completely emitting a certain amount of liquid (Nebulization time)as well as the TOR. The tests were performed with a liposomalformulation of amikacin.

TABLE 3 Properties of nebulizer membranes. Nebulization # of MMD timeTOR through Membrane (μm) (min) (g/min.) holes 26 1 (shown in FIG. 5with a 4.2 14.6 0.57 6,000 nozzle portion of 26 μm) 2 (shown in FIG. 6with a 4.3 9.3 0.89 6,000 nozzle portion of 19 μm) 3 (similar to FIG. 6)4.4 13.4 0.62 3,000 4 (similar to FIG. 6, 4.4 11.9 0.7 3,000 nozzleshorter than membrane 3)

Table 3 shows that the membrane 2 with the shorter nozzle portionprovides for an increased TOR and a reduced nebulization time by 5.3minutes, which is approximately 36% less as compared to the membrane 1.Table 3 also shows that the MMD did not vary significantly for eachmembrane tested. This is in contrast to the differences in TOR observedfor each membrane. Thus, in one embodiment, the nebulization time forthe nebulizer described herein is reduced significantly as compared toprior art nebulizers, without affecting the droplet size, as measured byMMD.

In addition to the membrane shown in FIGS. 5 and 6, membranes weremanufactured having the nozzle portion further reduced, and with 3,000through holes 26 (membranes 3 and 4, Table 3). In particular, a membrane3 was laser-drilled with a shorter nozzle portion, whereas membrane 4was manufactured using a shorter nozzle portion than membrane 3. Table 3indicates that even with 3,000 holes (membrane 3 and 4) a reduction inthe length of the nozzle portion results in an increased TOR compared tomembrane 1 with 6,000 holes. The comparison of the membrane 3 and 4 ascompared to the membrane 2 further shows that a combination of a highernumber of holes (6,000 as compared to 3,000) and a reduced length of thenozzle portion increases the TOR for the nebulizer.

In one embodiment, it is advantageous to use a laser drilling process ascompared to electroforming for manufacturing the through holes. Thethrough holes shown in FIGS. 5 and 6, manufactured by laser drilling,are substantially cylindrical or conical as compared to thefunnel-shaped entrance and exit of electro-formed through holes, e.g.,as disclosed in WO 01/18280. The vibration of the membrane, that is itsvibration velocity, may be transferred to the pharmaceutical formulationover a larger area by means of friction when the through holes aresubstantially cylindrical or conical as compared to the funnel-shapedentrance and exit of electro-formed through holes. The pharmaceuticalformulation, because of its own inertia, is then ejected from the exitopenings of the through holes resulting in liquid jets collapsing toform the aerosol. Without wishing to be bound by theory, it is thoughtthat because an electro-formed membrane comprises extremely bentsurfaces of the through holes, the surface or area for transferring theenergy from the membrane to the liquid is reduced.

However, the present invention may also be implemented in electro-formedmembranes, wherein the nozzle portion is defined by the continuousportion of the through hole in the extension direction starting from thesmallest diameter of the through hole towards the first side until itreaches a diameter 2× or 3× of the smallest diameter of the hole. In oneembodiment, the total length of the through hole is measured from thesmallest diameter to the first side.

Referring again to FIG. 1, so that the patient does not have to removeor to put down the therapeutic device from his mouth after inhaling theaerosol, the mouthpiece 3 has an opening 6 sealed by an elastic valveelement 7 (exhalation valve). If the patient exhales into the mouthpiece3 and hence into the nebulizing chamber 2, the elastic valve element 7opens so that the exhaled air is able to escape from the interior of thetherapeutic aerosol. On inhalation, ambient air flows through thenebulizing chamber 2. The nebulizing chamber 2 has an opening sealed(not shown) by a further elastic valve element (inhalation valve). Ifthe patient inhales through the mouthpiece 3 and sucks from thenebulizing chamber 2, the elastic valve element opens so that theambient air is able to enter into the nebulizing chamber and mixed withthe aerosol and leave the interior of the nebulizing chamber 2 to beinhaled. Further description of this process is provided in U.S. Pat.No. 6,962,151, which is incorporated by reference in its entirety forall purposes.

The nebulizer shown in FIG. 2 comprises a cylindrical storage vessel 10to supply a liquid that is fed to the membrane 5. As shown in FIG. 2,the oscillating membrane 5 may be arranged in an end wall 12 of thecylindrical liquid reservoir 10 to ensure that the liquid poured intothe liquid reservoir comes into direct contact with the membrane 5 whenthe aerosol generator is held in the position shown in FIG. 1. However,other methods may also be used to feed the liquid to the oscillatingmembrane without any change being necessary to the design of the deviceaccording to the invention for the generation of a negative pressure inthe liquid reservoir.

On the side facing the end wall 12, the cylindrical liquid container 10is open. The opening is used to pour the liquid into the liquidreservoir 10. Slightly below the opening on the external surface 13 ofthe peripheral wall 14 there is a projection 15 which serves as asupport when the liquid container is inserted in an appropriatelyembodied opening in a housing 35.

The open end of the liquid container 10 is closed by a flexible sealingelement 16. The sealing element 16 lies on the end of the peripheralwall 14 of the liquid container 10 and extends in a pot-shaped way intothe interior of the liquid container 10 whereby a conically running wallsection 17 is formed in the sealing element 16 and closed off by a flatwall section 18 of the sealing element 16. As discussed further below,forces act via the flat wall section 18 on the sealing element 16 and soin one embodiment the flat wall section 18 is thicker than the othersections of the sealing element 16. On the perimeter of the flat wallsection 18, there is a distance to the conical wall section 17 so thatthe conical wall section 17 may be folded when the flat wall section 18is moved upwards, relative to the representation in FIG. 2.

On the side of the flat wall section 18 facing away from the interior ofthe liquid container, there is a projection comprising a truncated conesection 19 and a cylindrical section 20. This design enables theprojection to be introduced and latched into an opening adapted to matchthe cylindrical section since the flexible material of the sealingelement 16 permits the deformation of the truncated cone section 19.

In one embodiment, the aerosol generator 4 comprises a slidable sleeve21 equipped with an opening of this type which is substantially a hollowcylinder open on one side. The opening for the attachment of the sealingelement 16 is embodied in an end wall of the slidable sleeve 21. Whenthe truncated cone 19 has latched into place, the end wall of theslidable sleeve 21 containing the opening lies on the flat sealingelement wall section 18. The latching of the truncated cone 19 into theslidable sleeve enables forces to be transmitted from the slidablesleeve 21 onto the flat wall section 18 of the sealing element 16 sothat the sealing section 18 follows the movements of the slidable sleeve21 in the direction of the central longitudinal axis of the liquidcontainer 10.

In a generalized form, the slidable sleeve 21 may be seen as a slidableelement, which may, for example, also be implemented as a slidable rodwhich may be stuck-on or inserted in a drill hole. Characteristic of theslidable element 21 is the fact that it may be used to apply asubstantially linearly directed force onto the flat wall element 18 ofthe sealing element 16. Overall, the decisive factor for the mode ofoperation of the aerosol generator according to the invention is thefact that a slidable element transmits a linear movement onto thesealing element so that an increase in volume occurs within the liquidreservoir 10. Since the liquid reservoir 10 is otherwise gas-tight, thiscauses a negative pressure to be generated in the liquid reservoir 10.

The sealing element 16 and the slidable element 21 may be produced inone piece, i.e., in one operation, but from different materials. Theproduction technology for this is available so that a one-piececomponent for the nebulizer is created, e.g., in a fully automaticproduction step.

In one embodiment, the slidable sleeve 21 is open on the end facing thedrill hole for the truncated cone but at least two diametricallyopposite lugs 22 and 23 protrude radially into the interior of theslidable sleeve 21. A collar 24 encircling the slidable sleeve extendsradially outwards. While the collar 24 is used as a support for theslidable sleeve 21 in the position shown in FIG. 5, the projections 22and 23 protruding into the interior of the slidable sleeve 21 are usedto absorb the forces acting on the slidable sleeve 21 in particularparallel to the central longitudinal axis. In one embodiment, theseforces are generated by means of two spiral grooves 25 which are locatedon the outside of the peripheral wall of a rotary sleeve 26.

In one embodiment, the nebulizer may be implemented with one of theprojections 22 or 23 and one groove 25. In a further embodiment, auniformly distributed arrangement of two or more projections and acorresponding number of grooves is provided.

In one embodiment, the rotary sleeve 26 is also a cylinder open on oneside whereby the open end is arranged in the slidable sleeve 21 and ishence facing the truncated cone 19 enabling the truncated cone 19 topenetrate the rotary sleeve 26. In addition, the rotary sleeve 26 isarranged in the slidable sleeve 21 in such a way that the projections 22and 23 lie in the spiral grooves 25. The inclination of the spiralgroove 25 is designed so that, when the rotary sleeve 26 is rotated inrelation to the slidable sleeve 21, the projections 22 and 23 slidealong the spiral grooves 25 causing a force directed parallel to thecentral longitudinal axis to be exerted on the sliding projections 22and 23 and hence on the slidable sleeve 21. This force displaces theslidable sleeve 21 in the direction of the central longitudinal axis sothat the sealing element 16 which is latched into the slidable sleeve'sdrill hole by means of the truncated cone is also substantiallydisplaced parallel to the central longitudinal axis.

The displacement of the sealing element 16 in the direction of thecentral longitudinal axis of the liquid container 10 generates anegative pressure in the liquid container 10, determined inter alia bythe distance by which the slidable sleeve 21 is displaced in thedirection of the central longitudinal axis. The displacement causes theinitial volume V_(RI) of the gas-tight liquid container 10 to increaseto the volume V_(RN) and thereby a negative pressure to be generated.The displacement is in turn defined by the design of the spiral grooves25 in the rotary sleeve 26. In this way, the aerosol generator accordingto the invention ensures that the negative pressure in the liquidreservoir 10 may be generated in the relevant areas by means of simplestructural measures.

To ensure that the forces to be applied to generate the negativepressure when handling the device remain low, the rotary sleeve 26 isembodied in one piece with a handle 27 whose size is selected to enablethe user to rotate the handle 27, and hence the rotary sleeve 26,manually without great effort. The handle 27 substantially has the shapeof a flat cylinder or truncated cone which is open on one side so that aperipheral gripping area 28 is formed on the external periphery of thehandle 27 which is touched by the user's hand to turn the handle 27.

Due to the design of the spiral grooves 25 and the overall comparativelyshort distance to be travelled by the slidable sleeve 21 in thelongitudinal direction to generate a sufficient negative pressure, inone embodiment, it is sufficient to turn the handle 27 and hence therotary sleeve 26 through a comparatively small angle of rotation. In oneembodiment, the angle of rotation lies within a range from 45 to 360degrees. This embodiment allows for the ease of handling of the deviceaccording to the invention and the therapeutic aerosol generatorequipped therewith.

In order to create a unit which may be operated simply and uniformlyfrom the slidable sleeve 21 and the rotary sleeve 26 including thehandle 27, in one embodiment, the aerosol generator described here has abearing sleeve 29 for bearing the slidable sleeve 21, whichsubstantially comprises a flat cylinder open on one side. The diameterof the peripheral wall 30 of the bearing sleeve 29 is smaller than theinternal diameter of the handle 27 and, in the example of an embodimentdescribed, is aligned on the internal diameter of a cylindrical latchingring 31 which is provided concentrically to the gripping area 28 of thehandle 27 but with a smaller diameter on the side of the handle 27 onwhich the rotary sleeve 26 is also arranged. Embodied on the side of thecylindrical latching ring 31 facing the rotary sleeve is a peripherallatching edge 32 which may be brought into engagement with latching lugs33 situated at intervals on the peripheral wall 30 of the bearing sleeve29. This allows the handle 27 to be located on the bearing sleeve 29whereby, as shown in FIG. 5, the handle 27 is placed on the open end ofthe bearing sleeve 29 and the latching edge 32 is interlatched with thelatching lugs 33.

To hold the slidable sleeve 21, an opening is provided in the centre ofthe sealed end of the bearing sleeve 29 in which the slidable sleeve 21is arranged, as may be identified in FIG. 2. The collar 24 of theslidable sleeve 21 lies in the position shown in FIG. 2 on the surfaceof the end wall of the bearing sleeve 29 facing the handle. Extendinginto the bearing opening are two diametrically opposite projections 51and 52, which protrude into two longitudinal grooves 53 and 54 on theperipheral surface of the slidable sleeve 21. The longitudinal grooves53 and 54 run parallel to the longitudinal axis of the slidable sleeve21. The guide projections 51 and 52 and the longitudinal grooves 53 and54 provide anti-rotation locking for the slidable sleeve 21 so that therotational movement of the rotary sleeve 26 results not in rotation butin the linear displacement of the slidable sleeve 21. As is evident fromFIG. 2, this ensures that the slidable sleeve 21 is held in thecombination of the handle 27 and the bearing sleeve 29 in an axiallydisplaceable way but locked against rotation. If the handle 27 isrotated in relation to the bearing sleeve 29, the rotary sleeve 26 alsorotates in relation to the slidable sleeve 21 whereby the slidingprojections 22 and 23 move along the spiral grooves 25. This causes theslidable sleeve 21 to be displaced in an axial direction in the openingof the bearing sleeve 29.

It is possible to dispense with the guide projections 51 and 52 in thebearing opening and the longitudinal grooves 53 and 54 in the slidablesleeve 21. In one embodiment, the guide projections 51 and 52 and thelongitudinal grooves 53 and 54 are not present in the aerosol generator,and the truncated cone 19, the cylinder sections 20 of the sealingelements 16 and the large-area support for the slidable sleeve 21holding the truncated cone on the flat sealing element section 18achieves anti-rotation locking of the slidable sleeve 21 by means offriction. In a further embodiment, the sealing element 16 is fixed so itis unable to rotate in relation to the bearing sleeve 29.

In one embodiment, provided on the surface of the sealed end of thebearing sleeve 19 facing away from the handle, is an annular firstsealing lip 34 concentric to the opening holding the slidable sleeve.The diameter of the first sealing lip 34 corresponds to the diameter ofthe peripheral wall 14 of the liquid container 10. As provided in FIG.2, this ensures that the first sealing lip 34 presses the sealingelement 16 on the end of the peripheral wall against the liquidreservoir 10 in such a way that the liquid reservoir 10 is sealed. Inaddition, the first sealing lip 34 may also fix the sealing element 16so that it is unable to rotate in relation to the liquid reservoir 10and the bearing sleeve 29. In one embodiment, excessive force need notbe applied in order to ensure that the aforesaid components of thedevice are unable to rotate in relation to each other.

In one embodiment, the forces required are generated at least to someextent by means of an interaction between the handle 27 and the housing35 in which the pharmaceutical formulation reservoir is embodied as onepiece or in which the pharmaceutical formulation (liquid) reservoir 10is inserted as shown in FIG. 2. In this case, the pharmaceuticalformulation reservoir 10 inserted in the casing with the peripheralprojection 15 lies at intervals on a support 36 in the housing 35 whichextends radially into the interior of the housing 35. This allows theliquid reservoir 10 to be easily removed from the housing 35 forpurposes of cleaning. In the embodiment shown in FIG. 2, support is onlyprovided at intervals, and therefore, openings are provided for ambientair when the patient inhales, described in more detail below.

Identifiable in FIG. 2 is the rotary lock, which is implemented by meansof the handle 27 on the one hand and the housing 35 on the other. Shownare the locking projections 62 and 63 on the housing 35. However, thereare no special requirements with regard to the design of the rotary lockas far as the device according to invention is concerned for thegeneration of the negative pressure in the liquid reservoir 10.

In one embodiment, the liquid reservoir 10 is configured to have avolume V_(RN) of at least at least 16 mL, at least about 16 mL, at least18 mL, at least about 18 mL, at least 20 mL or at least about 20 mL sothat when for example, an amount of 8 mL of liquid (e.g., aminoglycosidepharmaceutical formulation) to be emitted in the form of an aerosol iscontained in (filled or poured into) the liquid reservoir 10, an aircushion of 8 mL or about 8 mL is provided. That is, the ratio of thevolume V_(RN) to the initial volume of liquid V_(L) within the liquidreservoir 10 is at least 2.0 and the ratio between the volume V_(A) of agas and V_(L) of the liquid is at least 1.0. It has been shown that aliquid reservoir having a volume V_(RN) of about 15.5 mL, about 19.5 mLand about 22.5 mL are efficient, and that efficiency increases with theincrease in V_(RN).

In one embodiment, the ratio between V_(RN) and V_(L) is at least 2.0,at least about 2.0, at least 2.4, at least about 2.4, at least 2.8 or atleast about 2.8. In one embodiment, the ratio between V_(A) and V_(L) isat least 1.0, at least 1.2, at least 1.4, at least 1.6 or at least 1.8.In another embodiment, the ratio between V_(A) and V_(L) is at leastabout 1.0, at least about 1.2, at least about 1.4, at least about 1.6 orat least about 1.8.

The volume of the air cushion, in one embodiment, is at least 2 mL, atleast about 2 mL, at least 4 mL, at least about 4 mL, is at least 6 mL,at least about 6 mL, at least 8 mL, at least about 8 mL, at least 10 mL,at least about 10 mL, at least 11 mL, at least about 11 mL, at least 12mL, at least about 12 mL, at least 13 mL, at least about 13 mL, at least14 mL or at least about 14 mL. In one embodiment, the volume of the aircushion is at least about 11 mL or at least about 14 mL. In oneembodiment, the volume of the air cushion is from about 6 mL to about 15mL, and the ratio between V_(RN) and V_(L) is at least about 2.0 to atleast about 3.0. In a further embodiment, the between V_(RN) and V_(L)is at least about 2.0 to about at least about 2.8.

The volume of the air cushion, in one embodiment, is about 2 mL, about 4mL, about 6 mL, about 8 mL, about 10 mL, about 11 mL, about 12 mL, about13 mL, or about 14 mL.

In one embodiment, the ratio of the volume V_(RN) to the initial volumeof liquid V_(L) is at least 2.0. Theoretically an unlimited enlargementof the increased volume V_(RN) of the liquid reservoir 10 will result ina nearly stable negative pressure range. In one embodiment, the ratio ofthe volume V_(RN) to the initial volume of liquid V_(L) is within therange between 2.0 and 4.0 and in a further embodiment is between 2.4 and3.2. Two examples of the ratio ranges (V_(RN)/V_(L)) for differentinitial volume of liquid V_(L) between 4 mL and 8 mL are given in Table4, below.

TABLE 4 Nebulizer Reservoir specifications V_(L) V_(RN) Ratio(V_(RN)/V_(L)) 4 mL  8.0-16.0 2.0-4.0 4 mL  9.5-12.8 2.4-3.2 5 mL10.0-20.0 2.0-4.0 5 mL 12.0-16.0 2.4-3.2 6 mL 12.0-24.0 2.0-4.0 6 mL14.5-19.2 2.4-3.2 8 mL 16.0-32.0 2.0-4.0 8 mL 19.5-25.6 2.4-3.2

The systems provided herein may be used to treat a variety of pulmonaryinfections in subjects in need thereof. Among the pulmonary infections(such as in cystic fibrosis patients) that can be treated with themethods of the invention are gram negative infections. In oneembodiment, infections caused by the following bacteria are treatablewith the systems and formulations provided herein: Pseudomonas (e.g., P.aeruginosa, P. paucimobilis, P. putida, P. fluorescens, and P.acidovorans), Burkholderia (e.g., B. pseudomallei, B. cepacia, B.cepacia complex, B. dolosa, B. fungorum, B. gladioli, B. multivorans, B.vietnamiensis, B. pseudomallei, B. ambifaria, B. andropogonis, B.anthina, B. brasilensis, B. caledonica, B. caribensis, B. caryophylli),Staphylococcus (e.g., S. aureus, S. auricularis, S. carnosus, S.epidermidis, S. lugdunensis), Methicillin-resistant Staphylococcusaureus (MRSA), Streptococcus (e.g., Streptococcus pneumoniae),Escherichia coli, Klebsiella, Enterobacter, Serratia, Haemophilus,Yersinia pestis, Mycobacterium, nontuberculous Mycobacterium (e.g., M.avium, M. avium subsp. hominissuis (MAH), M. abscessus, M. chelonae, M.bolletii, M. kansasii, M. ulcerans, M. avium, M. avium complex (MAC) (M.avium and M. intracellulare), M. conspicuum, M. kansasii, M peregrinum,M. immunogenum, M. xenopi, M. marinum, M. malmoense, M. marinum, M.mucogenicum, M. nonchromogenicum, M. scrofulaceum, M. simiae, M.smegmatis, M. szulgai, M. terrae, M. terrae complex, M. haemophilum, M.genavense, M. asiaticum, M. shimoidei, M. gordonae, M. nonchromogenicum,M. triplex, M. lentiflavum, M. celatum, M. fortuitum, M. fortuitumcomplex (M. fortuitum and M. chelonae)).

In one embodiment, the systems described herein are used to treat aninfection caused by a nontuberculous mycobacterial infection. In oneembodiment, the systems described herein are used to treat an infectioncaused by Pseudomonas aeruginosa, Mycobacterium abscessus, Mycobacteriumavium or M. avium complex. In a further embodiment, a patient withcystic fibrosis is treated for a Pseudomonas aeruginosa, Mycobacteriumabscessus, Mycobacterium avium, or Mycobacterium avium complex infectionwith one or more of the systems described herein. In even a furtherembodiment, the Mycobacterium avium infection is Mycobacterium avium subsp. hominissuis.

In one embodiment, a patient with cystic fibrosis is treated for apulmonary infection with one of the systems provided herein. In afurther embodiment, the pulmonary infection is a Pseudomonas infection.In yet a further embodiment, the Pseudomonas infection is P. aeruginosa.In a further embodiment, the aminoglycoside in the system is amikacin.

In one embodiment, the system provided herein is used for the treatmentor prophylaxis of Pseudomonas aeruginosa, Mycobacterium abscessus,Mycobacterium avium or Mycobacterium avium complex lung infection in acystic fibrosis patient or a non-cystic fibrosis patient. In a furtherembodiment, the system provided herein comprises a liposomalaminoglycoside formulation. In a further embodiment, the aminoglycosideis selected from amikacin, apramycin, arbekacin, astromicin,capreomycin, dibekacin, framycetin, gentamicin, hygromycin B,isepamicin, kanamycin, neomycin, netilmicin, paromomycin,rhodestreptomycin, ribostamycin, sisomicin, spectinomycin, streptomycin,tobramycin, verdamicin or a combination thereof. In even a furtherembodiment, the aminoglycoside is amikacin, e.g., amikacin sulfate.

An obstacle to treating infectious diseases such as Pseudomonasaeruginosa, the leading cause of chronic illness in cystic fibrosispatients is drug penetration within the sputum/biofilm barrier onepithelial cells (FIG. 7). In FIG. 7, the donut shapes representliposomal/complexed aminoglycoside, the “+” symbol represents freeaminoglycoside, the “−” symbol mucin, alginate and DNA, and the solidbar symbol represents Pseudomonas aeruginosa. This barrier comprisesboth colonized and planktonic P. aeruginosa embedded in alginate orexopolysaccharides from bacteria, as well as DNA from damagedleukocytes, and mucin from lung epithelial cells, all possessing a netnegative charge. The negative charge binds up and prevents penetrationof positively charged drugs such as aminoglycosides, rendering thembiologically ineffective (Mendelman et al., 1985). Without wishing to bebound by theory, entrapment of aminoglycosides within liposomes or lipidcomplexes shields or partially shields the aminoglycosides fromnon-specific binding to the sputum/biofilm, allowing for liposomes orlipid complexes (with entrapped aminoglycoside) to penetrate (FIG. 7).

In another embodiment, a patient is treated for nontuberculousmycobacteria lung infection with one of the systems provided herein. Ina further embodiment, the system provided herein comprises a liposomalamikacin formulation.

In another embodiment, the system provided herein is used for thetreatment or prophylaxis of one or more bacterial infections in a cysticfibrosis patient. In a further embodiment, the system provided hereincomprises a liposomal aminoglycoside formulation. In a furtherembodiment, the aminoglycoside is amikacin.

In another embodiment, the system provided herein is used for thetreatment or prophylaxis of one or more bacterial infections in apatient with bronchiectasis. In a further embodiment, the systemprovided herein comprises a liposomal aminoglycoside formulation. In afurther embodiment, the aminoglycoside is amikacin or amikacin sulfate.

In yet another embodiment, the system provided herein is used for thetreatment or prophylaxis of Pseudomonas aeruginosa lung infections innon-CF bronchiectasis patients. In a further embodiment, the systemprovided herein comprises a liposomal aminoglycoside formulation. In afurther embodiment, the aminoglycoside is amikacin.

As provided herein, the present invention provides aminoglycosideformulations administered via inhalation. In one embodiment, the MMAD ofthe aerosol is about 3.2 μm to about 4.2 μm, as measured by the AndersonCascade Impactor (ACI), or about 4.4 μm to about 4.9 μm, as measured bythe Next Generation Impactor (NGI).

In one embodiment, the nebulization time of an effective amount of anaminoglycoside formulation provided herein is less than 20 minutes, lessthan 18 minutes, less than 16 minutes or less than 15 minutes. In oneembodiment, the nebulization time of an effective amount of anaminoglycoside formulation provided herein is less than 15 minutes orless than 13 minutes. In one embodiment, the nebulization time of aneffective amount of an aminoglycoside formulation provided herein isabout 13 minutes.

In one embodiment, the formulation described herein is administered oncedaily to a patient in need thereof.

EXAMPLES

The present invention is further illustrated by reference to thefollowing Examples. However, it should be noted that these Examples,like the embodiments described above, are illustrative and are not to beconstrued as restricting the scope of the invention in any way.

Example 1 Comparison Of Nebulizer Reservoir Volumes

In this example, the aerosol generator was an investigational eFlow®nebulizer, modified for use with liposomal aminoglycoside formulationsprovided herein, of Pari Pharma GmbH, Germany. A first aerosol generatorhad an initial volume of the liquid reservoir V_(RI) of 13 mL (A), asecond one of 17 mL (B), a third one of 22 mL (C) and a fourth one of 20mL (D). That is the increased volume V_(RN) of the first one had 15.5mL, the second one 19.5 mL, the third 24.5 mL and the fourth 22.5 mL.

8 mL of a liposomal amikacin formulation was poured into the liquidreservoir 10. As shown in FIG. 8, an air cushion of 8 mL resulted in anaerosol generation time period upon complete emission of 8 mL of theformulation in the liquid reservoir of between 14 and 16 minutes. An aircushion of 12 mL, however, decreased the aerosol generation time to arange between 12 and approximately 13 minutes. The air cushion of 17 mLfurther decreases the aerosol generation time to an amount between 10and 12 minutes (FIG. 6).

Further, the first (A) and third (C) version of the aerosol generatorhad been used together with 8 mL of the liposomal amikacin formulation.An initial negative pressure of equal to or less than 50 mbar wasgenerated within the liquid reservoir. In addition, the negativepressure was measured during the aerosol generation and is shown overthe aerosol generation time in FIG. 9. In other words, FIG. 9 showsexperimental data comparing the negative pressure range during theaerosol generation time for a liquid reservoir (C) having a volumeV_(RN) of 24.5 mL and a liquid reservoir (A) having a volume V_(RN) of15.5 mL. The initial amount of amikacin formulation V_(L) was 8 mL andthe initial negative pressure was about 50 mbar. The graph indicatesthat a larger air cushion prevents the negative pressure from increasingabove a critical value of 300 mbar.

The dependence of aerosol generator efficiency (proportional to liquidoutput rate or total output rate) on different negative pressures wasmeasured with the nebulizer described above. A liposomal amikacinformulation having a viscosity in the range of 5.5 to 14.5 mPa×s atsheer forces between 1.1 and 7.4 Pa (thixotrope) was used in theexperiment. As shown in FIG. 10, the efficiency is optimum in a negativepressure range between 150 mbar and 300 mbar. As also shown in FIG. 10,the efficiency decreases at a negative pressure below approximately 150mbar and at a negative pressure of above 300 mbar.

Furthermore, the same liposomal amikacin formulation as in FIG. 8 wasused in four different aerosol generators based on the modified eFlow®,wherein the first aerosol generator (A) is a modified eFlow® with anincreased volume V_(RN) of the liquid reservoir of 19.5 mL and filledwith 8 mL of the liposomal amikacin formulation.

The second aerosol generator (B) had a reservoir with an increasedvolume V_(RN) of 16 mL filled with 8 mL of the mentioned liposomalamikacin formulation, the third aerosol generator (C) one had anincreased volume V_(RN) of 24.5 mL, filled with 8 mL of the mentionedliquid. The fourth aerosol generator had an increased volume V_(RN) ofthe liquid reservoir of 22.5 mL, and was filled with 8 mL of theaforementioned liposomal amikacin formulation.

FIG. 11 shows experimental data of these four aerosol generators filledwith 8 mL of the liposomal amikacin formulation. The results show theaerosol generation time for complete emission of the liposomal amikacinformulation within the liquid reservoir in relation to the ratio of theincreased volume of the liquid reservoir (V_(RN)) to the initial volumeof liquid in the liquid reservoir before use (V_(L)). FIG. 11 indicatesthat with the modified aerosol generator device (A) an aerosolgeneration time of approximately 16 minutes was required, whereas theaerosol generation time decreased with an increased ratio V_(RN)/V_(L).The data also shows that the aerosol generation time could be reduced byapproximately 4 minutes to below 12 minutes with the third aerosolgenerator device (C).

The data provided in Example 1 therefore indicates that a larger aircushion enables the operation of the aerosol generator for a longer timein an efficient negative pressure range so that the total aerosolgeneration time may significantly be reduced. Therefore, even largeamounts of liquid such as 8 mL may be nebulized (emitted in form ofaerosol) in a period of time below 12 minutes.

Example 2 Aerosol Properties of Amikacin Formulation

Eleven different lots of the liposomal amikacin formulation wereexamined with the modified eFlow® nebulizer (i.e., modified for use withthe liposomal aminoglycoside formulations described herein) having amodified 40 mesh membrane fabricated as described herein, and areservoir with an 8 mL liquid capacity and aforementioned air cushion.Cascade impaction was performed using either the ACI (Anderson CascadeImpactor) or the NGI (Next Generation Impactor) to establish aerosolproperties: mass median aerodynamic diameter (MMAD), geometric standarddeviation (GSD), and Fine-Particle-Fraction (FPF).

Mass Median Aerodynamic Diameter (MMAD) Measurement with ACI

An Anderson Cascade Impactor (ACI) was used for MMAD measurements andthe nebulization work was conducted inside a ClimateZone chamber(Westech Instruments Inc., GA) to maintain temperature and relativehumanity % during nebulization. The ClimateZone was pre-set to atemperature of 18° C. and a relative humanity of 50%. The ACI wasassembled and loaded inside the ClimateZone. A probe thermometer (VWRdual thermometer) was attached to the surface of ACI at stage 3 tomonitor the temperature of ACI. Nebulization was started when thetemperature of the ACI reached 18±0.5° C.

With the 8 mL handsets loaded with 8 mL, it was found that the ACI couldnot handle the whole 8 mL dose; i.e., amikacin liposomal formulationdeposited on ACI plate 3 overflowed. It was determined that the percentdrug distribution on each ACI stage was not affected by the amount ofliposomal amikacin formulation collected inside the ACI as long as therewas no liquid overflow at ACI stage 3 (data not shown). Therefore fornebulization, the nebulizer was either filled with 4 mL liposomalamikacin formulation and nebulized until empty or filled with 8 mL ofliposomal amikacin formulation and nebulized for about 6 minutes ofcollection time (i.e., ˜4 mL).

The nebulizate was collected at a flow rate of 28.3 L/min in the ACIwhich was cooled to 18° C. The nebulization time was recorded and thenebulization rate calculated based on the difference in weight (amountnebulized) divided by the time interval.

After the nebulizate was collected, ACI collection plates 0, 1, 2, 3, 4,5, 6 and 7 were removed, and each was loaded into its own petri dish. Anappropriate amount of extraction solution (20 mL for plates 2, 3, and 4,and 10 mL for plates 0, 1, 5, 6, and 7) was added to each Petri dish todissolve the formulation deposited on each plate. Samples from plates 0,1, 2, 3, 4, 5 and 6 were further diluted appropriately with mobile phaseC for HPLC analysis. Sample from plate 7 was directly analyzed by HPLCwithout any further dilution. The ACI Filter was also transferred to a20 mL vial and 10 mL extraction solution was added, and the capped vialvortexed to dissolve any formulation deposited on it. Liquid samplesfrom the vial were filtered (0.2 μm) into HPLC vials for HPLC analysis.The induction port with connector was also rinsed with 10 mL extractionsolution to dissolve the formulation deposited on it, and the sample wascollected and analyzed by HPLC with 2 time dilution. Based on theamikacin amount deposited on each stage of the impactor, mass medianaerodynamic diameter (MMAD), geometric standard deviation (GSD) and fineparticle fraction (FPF) were calculated.

In the cases for nebulizers loaded with 8 mL and nebulized for 6 minutesfine particle dose (FPD) was normalized to the volume of formulationnebulized in order to compare FPD across all experiments. FPD(normalized to the volume of formulation nebulized) was calculatedaccording to the following equation:

${F\; P\; D\mspace{14mu}\left( {{normalized}\mspace{14mu}{to}\mspace{14mu}{volume}\mspace{14mu}{nebulized}} \right)\mspace{11mu}\left( \frac{mg}{mL} \right)} = \frac{{Amikacin}\mspace{14mu}{Recovered}_{ACI} \times F\; P\; F\mspace{14mu}({mg})}{{Arikace}\mspace{14mu}{Nebulized}\mspace{11mu}{(g) \div {{Density}\left( \frac{g}{mL} \right)}}}$

Mass Median Aerodynamic Diameter (MMAD) Measurement with NGI

A Next Generation Impactor (NGI) was also used for MMAD measurements andthe nebulization work was conducted inside a ClimateZone chamber(Westech Instruments Inc., GA) to maintain temperature and RH % duringnebulization. The ClimateZone was pre-set to a temperature of 18° C. anda relative humanity of 50%. The NGI was assembled and loaded inside theClimateZone. A probe thermometer (VWR dual thermometer) was attached tothe surface of NGI to monitor the temperature of NGI. Nebulization wasstarted when the temperature of the NGI reached 18±0.5° C.

8 mL of the liposomal amikacin formulation was added to the nebulizerand nebulized. When there was no more aerosol observed, the timer wasstopped. The nebulizate was collected at a flow rate of 15 L/min in theNGI which was cooled to 18° C. The nebulization time was recorded andthe nebulization rate calculated based on the difference in weight(amount nebulized) divided by the time interval.

After aerosol collection was done, the NGI tray with tray holder wasremoved from NGI. An appropriate amount of extraction solution was addedto NGI cups 1, 2, 3, 4, 5, 6, 7 and MOC to dissolve the formulationdeposited on these cups. This material was transferred to a volumetricflask respectively. For NGI cups 1, 2, and 6, 25 ml volumetric flaskswere used; for NIG cups 2, 3, 4, 50 ml volumetric flasks were used. Moreextraction solution was added to the cups and again transferred to thevolumetric flask. This procedure was repeated several times in order totransfer formulation deposited on the NGI cup to the volumetric flaskcompletely. The volumetric flasks were topped up to bring the finalvolume to either 25 ml or 50 ml and shaken well before sampled. Samplesfrom cups 1, 2, 3, 4, 5, 6 and 7 were further diluted appropriately withmobile phase C for HPLC analysis. Sample from MOC was directly analyzedby HPLC without any further dilution. The NGI Filter was alsotransferred to a 20 mL vial and 10 mL extraction solution was added, andthe capped vial vortexed to dissolve any formulation deposited on it.Liquid samples from the vial were filtered (0.2 micron) into HPLC vialsfor HPLC analysis. The Induction port with connector was also rinsedwith 10 mL extraction solution to dissolve the formulation deposited onit, and the sample was collected and analyzed by HPLC with 11 timedilution.

Based on the amikacin amount deposited on each stage of the impactor,MMAD, GSD and FPF were calculated.

FPD was normalized to the volume of formulation nebulized in order tocompare FPD across all experiments. FPD (normalized to the volume offormulation nebulized) was calculated according to the followingequation:

${F\; P\; D\mspace{14mu}\left( {{normalized}\mspace{14mu}{to}\mspace{14mu}{volume}\mspace{14mu}{nebulized}} \right)\mspace{11mu}\left( \frac{mg}{mL} \right)} = \frac{{Amikacin}\mspace{14mu}{Recovered}_{ACI} \times F\; P\; F\mspace{14mu}({mg})}{{Arikace}\mspace{14mu}{Nebulized}\mspace{11mu}{(g) \div {{Density}\left( \frac{g}{mL} \right)}}}$

The results of these experiments are provided in FIGS. 12 and 13 andTable 5, below.

TABLE 5 Aerosol Characteristics ACI APSD Data NGI APSD Data NebulizationData Aerosol Neb. FPF <5 Aerosol Neb. FPF <5 Aerosol Neb. % Assoc.Amikacin Head Rate MMAD μm Head Rate MMAD μm Head Rate Amikacin Conc.Run ID (g/min) (μm) GSD (%) ID (g/min) (μm) GSD (%) ID (g/min) Post-Neb66.9 1 J 0.66 3.7 1.7 70.4 J 0.68 4.7 1.7 55.5 A 0.63 69.1 mg/mL 2 K0.62 3.7 1.7 71.7 K 0.63 4.5 1.7 57.5 B 0.60 68.2 3 L 0.65 4.0 1.8 66.1L 0.71 4.8 1.7 52.6 C 0.56 69.9 70.8 1 M 0.64 3.9 1.7 67.1 M 0.74 4.71.7 54.8 M 0.65 64.5 mg/mL 2 N 0.68 4.0 1.8 64.8 N 0.78 4.9 1.7 52.0 N0.67 66.3 3 O 0.69 3.9 1.7 67.4 O 0.75 4.8 1.7 52.7 O 0.64 69.4 64.6 1 C0.78 4.0 1.8 65.5 A 0.74 4.7 1.7 54.6 G 0.72 71.9 mg/mL 2 D 0.64 3.7 1.770.2 B 0.73 4.7 1.7 55.2 H 0.64 71.5 3 H 0.62 3.7 1.7 70.6 C 0.78 4.71.7 54.4 J 0.68 71.8 68.5 1 E 0.69 3.8 1.7 69.4 E 0.70 4.6 1.7 56.2 E0.60 69.1 mg/mL 2 F 0.78 4.0 1.8 66.2 F 0.83 4.7 1.7 54.8 F 0.67 70.4 3G 0.65 3.8 1.7 69.1 G 0.69 4.6 1.7 57.2 G 0.61 69.5 65.7 1 V 0.74 3.81.7 69.1 V 0.84 4.7 1.7 54.3 M 0.64 69.2 mg/mL 2 W 0.72 3.8 1.7 68.3 W0.78 4.7 1.7 55.1 N 0.74 67.9 3 X 0.70 3.9 1.7 68.0 X 0.74 4.7 1.7 54.5O 0.63 68.6 66.8 1 J 0.63 3.7 1.8 70.6 A 0.77 4.8 1.7 53.3 A 0.70 73.2mg/mL 2 K 0.59 3.7 1.8 70.4 D 0.55 4.7 1.7 55.1 B 0.70 72.4 3 L 0.64 3.91.8 66.6 H 0.65 4.7 1.7 55.3 C 0.83 72.8 69.2 1 S 0.66 3.8 1.7 68.6 U0.80 4.8 1.7 53.0 S 0.69 70.7 mg/mL 2 T 0.73 3.8 1.7 68.3 V 0.78 4.5 1.758.2 T 0.75 71.0 3 U 0.54 4.0 1.8 65.5 W 0.78 4.7 1.7 55.3 U 0.80 71.171.4 1 Q 0.66 3.8 1.7 68.4 M 0.75 4.6 1.7 56.7 P 0.71 72.4 mg/mL 2 R0.71 3.9 1.8 66.6 N 0.78 4.8 1.7 52.9 Q 0.68 70.0 3 S 0.66 3.8 1.7 68.5O 0.78 4.6 1.7 55.7 R 0.74 71.7 69.9 1 C 0.77 4.1 1.8 64.3 J 0.68 4.41.7 59.4 A 0.68 73.8 mg/mL 2 D 0.62 3.8 1.7 68.6 K 0.69 4.4 1.7 59.7 B0.63 73.6 3 H 0.61 3.7 1.7 70.3 L 0.77 4.7 1.7 55.6 C 0.70 75.7 72.2 1 T0.70 3.8 1.7 69.8 T 0.74 4.6 1.7 55.8 M 0.65 67.9 mg/mL 2 U 0.76 3.9 1.767.0 U 0.74 4.7 1.7 54.8 N 0.71 70.3 3 X 0.67 3.9 1.7 67.9 X 0.70 4.71.7 54.9 P 0.57 71.8 70.4 1 C 0.66 3.6 1.7 73.1 J 0.65 4.5 1.7 58.0 H0.59 60.1 mg/mL 2 D 0.57 3.5 1.7 74.1 K 0.65 4.5 1.7 59.1 J 0.69 59.3 3E 0.63 3.5 1.7 75.2 L 0.66 4.7 1.7 55.6 K 0.63 58.5

Example 3 Nebulization Rate Study

Nebulization rate studies (grams of formulation nebulized per minute)were conducted in a biosafety cabinet (Model 1168, Type B2, FORMAScientific). The assembled nebulizer (handset with mouth piece andaerosol head) was first weighed empty (W₁), then a certain volume offormulation was added and the nebulizer device was weighed again (W₂).The nebulizer and timer were started and the formulations nebulized werecollected in a chilled impinger at a flow rate of ˜8 L/min (see FIG. 14for details of experimental setup). When there was no more aerosolobserved, the timer was stopped. The nebulizer was weighed again (W₃),and the time of nebulization (t) was recorded. Total formulationnebulized was calculated as W₂−W₃ and total drug residue afternebulization was calculated as W₃−W₁. The nebulization rate offormulation was calculated according to the following equation:

${{Nebulization}\mspace{14mu}{Rate}\mspace{14mu}\left( \frac{g}{\min} \right)} = \frac{W_{2} - W_{3}}{t}$

Nebulization rates in g/min., as well as other related results, forliposomal amikacin nebulized using a nebulizer fabricated according tothe specification (twenty four aerosol heads were selected and were usedin these studies) are captured in Table 6.

TABLE 6 Formulation nebulization rates (g/min) Neb Formulation NebAerosol Time Nebulized Rate Run Head # (min) (g) (g/min) 1 1 11.907.7346 0.65 2 2 11.58 8.0573 0.70 3 3 10.87 8.0029 0.74 4 4 13.63 7.93590.58 5 5 12.60 8.0577 0.64 6 6 12.62 8.0471 0.64 7 7 14.23 8.073 0.57 88 14.67 8.0872 0.55 9 9 13.58 7.9235 0.58 10 10 12.28 7.9649 0.65 11 1112.33 8.1872 0.66 12 12 13.17 8.1694 0.62 13 1 11.22 7.9991 0.71 14 211.90 8.1392 0.68 15 3 12.17 8.0162 0.66 16 4 12.90 8.0174 0.62 17 511.22 7.893 0.70 18 6 10.23 8.0401 0.79 19 7 12.55 8.0988 0.65 20 814.88 7.8781 0.53 21 9 13.68 8.1678 0.60 22 10 12.33 8.2253 0.67 23 1112.60 8.0783 0.64 24 12 11.83 7.946 0.67 25 1 11.92 8.1703 0.69 26 211.95 7.9837 0.67 27 3 13.63 8.1536 0.60 28 4 11.90 7.9376 0.67 29 512.27 8.1727 0.67 30 6 12.27 8.0875 0.66 31 7 13.65 8.0767 0.59 32 815.80 8.1183 0.51 33 9 13.65 8.1373 0.60 34 10 12.98 7.8864 0.61 35 1111.63 8.1445 0.70 36 12 12.95 8.0232 0.62 37 13 12.80 7.9098 0.62 38 1410.25 8.0328 0.78 39 15 12.13 7.9911 0.66 40 16 12.33 8.1756 0.66 41 1712.47 7.9417 0.64 42 18 13.17 7.9046 0.60 43 19 13.92 7.5367 0.54 44 2011.47 8.1466 0.71 45 21 11.67 7.9366 0.68 46 22 13.17 8.0613 0.61 47 2312.77 7.8596 0.62 48 24 12.25 8.0552 0.66 49 13 13.67 7.9379 0.58 50 1410.55 8.0221 0.76 51 15 11.80 8.0555 0.68 52 16 10.08 8.1639 0.81 53 1711.08 7.9121 0.71 54 18 12.28 8.017 0.65 55 19 11.40 7.9415 0.70 56 2012.17 8.211 0.67 57 21 11.45 8.18 0.71 58 22 12.03 7.8946 0.66 59 2312.83 8.0771 0.63 60 24 11.97 7.9936 0.67 61 13 12.38 8.0054 0.65 62 1410.53 8.0492 0.76 63 15 11.82 7.8161 0.66 64 16 11.83 8.1169 0.69 65 1712.67 8.1778 0.65 66 18 12.03 8.2436 0.69 67 19 13.17 7.8821 0.60 68 2012.17 8.2397 0.68 69 21 11.78 8.1814 0.69 70 22 11.78 8.3443 0.71 71 2313.17 8.1699 0.62 72 24 11.50 8.0413 0.70 Average 12.4 ± 1.1 8.0 ± 0.10.66 ± 0.06

Example 4 Percent of Associate Amikacin Post-Nebulization and NebulizateCharacterization

The free and liposomal complexed amikacin in the nebulizate of Example 3was measured. As mentioned in Example 3, the nebulizate was collected ina chilled impinger at a flow rate of 8 L/min (FIG. 14).

The nebulizate collected in the impinger was rinsed with 1.5% NaCl andtransferred to a 100 mL or 50-mL volumetric flask. The impinger was thenrinsed several times with 1.5% NaCl in order to transfer all theformulation deposited in the impinger to the flask. To measure the freeamikacin concentration of the nebulizate, 0.5 mL of the dilutednebulizate inside the volumetric flask was taken and loaded to anAmicon® Ultra—0.5 mL 30K centrifugal filter device (regeneratedcellulose, 30K MWCO, Millipore) and this device was centrifuged at 5000G at 15° C. for 15 minutes. An appropriate amount of filtrate was takenand was diluted 51 times with mobile phase C solution. Amikacinconcentration was determined by HPLC. To measure total amikacinconcentration of the nebulizate, an appropriate amount of the dilutednebulizate inside the volumetric flask was taken and diluted (alsodissolved) 101 times in extraction solution (perfluoropentanoicacid:1-propanol:water (25:225:250, v/v/v)) and the amikacinconcentration determined by HPLC.

The percent associated amikacin post-nebulization was calculated by thefollowing equation:

${\%\mspace{14mu}{Associated}} = {\frac{{Concentration}_{\;{Total}} - {Concentration}_{\;{Free}}}{{Concentration}_{\;{Total}}} \times 100}$

The percent associated amikacin post-nebulization and total doserecovery from nebulization experiments described in Table 6 aresummarized in Table 7. Corresponding nebulization rates were alsoincluded in Table 7.

TABLE 7 Percent associated amikacin post-nebulization and total doserecovered Aerosol % Recovered Neb Rate Run Head # Associated % (g/min) 11 65.7 104 0.65 2 2 65.1 97 0.70 3 3 64.5 96 0.74 4 4 66.1 97 0.58 5 562.1 92 0.64 6 6 65.5 95 0.64 7 7 63.5 94 0.57 8 8 60.4 92 0.55 9 9 65.093 0.58 10 10 72.7 102 0.65 11 11 64.9 92 0.66 12 12 66.7 97 0.62 13 167.1 102 0.71 14 2 64.2 97 0.68 15 3 68.8 98 0.66 16 4 65.5 94 0.62 17 566.1 98 0.70 18 6 65.7 94 0.79 19 7 65.5 100 0.65 20 8 64.8 95 0.53 21 960.3 94 0.60 22 10 59.1 95 0.67 23 11 63.3 95 0.64 24 12 66.3 98 0.67 251 66.4 104 0.69 26 2 63.5 93 0.67 27 3 62.9 93 0.60 28 4 64.2 93 0.67 295 64.9 99 0.67 30 6 68.2 98 0.66 31 7 61.0 96 0.59 32 8 59.9 96 0.51 339 63.0 95 0.60 34 10 58.1 95 0.61 35 11 66.1 98 0.70 36 12 64.2 98 0.6237 13 65.6 100 0.62 38 14 68.9 96 0.78 39 15 63.7 97 0.66 40 16 64.7 970.66 41 17 69.1 97 0.64 42 18 70.2 94 0.60 43 19 61.2 93 0.54 44 20 63.491 0.71 45 21 67.7 99 0.68 46 22 66.7 96 0.61 47 23 67.2 93 0.62 48 2469.6 98 0.66 49 13 66.2 102 0.58 50 14 66.9 97 0.76 51 15 66.7 96 0.6852 16 64.7 96 0.81 53 17 65.1 96 0.71 54 18 67.6 98 0.65 55 19 66.7 970.70 56 20 63.6 99 0.67 57 21 68.1 101 0.71 58 22 64.8 99 0.66 59 2366.2 97 0.63 60 24 67.4 103 0.67 61 13 64.2 99 0.65 62 14 68.7 101 0.7663 15 66.0 100 0.66 64 16 67.7 103 0.69 65 17 66.4 100 0.65 66 18 66.298 0.69 67 19 68.3 100 0.60 68 20 67.9 101 0.68 69 21 67.1 98 0.69 70 2266.2 101 0.71 71 23 67.0 97 0.62 72 24 68.0 100 0.70 Average 65.5 ± 2.697 ± 3 0.66 ± 0.06

The total concentration of amikacin in the liposomal amikacinformulation was measured during this study with the rest of the samplesusing the same HPLC and amikacin standards. The value obtained was 64mg/mL amikacin. The % associated amikacin post-nebulization valuesranged from 58.1% to 72.7%, with an average value of 65.5±2.6%; for 8 mLliposomal amikacin formulation nebulized, the total recovered amount ofamikacin ranged from 426 mg to 519 mg, with an average value of 476±17mg; the calculated amount of amikacin nebulized (according to the weightof the liposomal amikacin formulation nebulized in Table 7) ranged from471 mg to 501 mg, with an average value of 490±8 mg; the total amikacinrecovery ranged from 91% to 104%, with an average value of 97±3% (n=72).

Liposome Size

The liposomal amikacin formulation (64 mg/mL amikacin), eitherpre-nebulized or post-nebulized, was diluted appropriately with 1.5%NaCl and the liposome particle size was measured by light scatteringusing a Nicomp 380 Submicron Particle Sizer (Nicomp, Santa Barbara,Calif.).

The liposome sizes post-nebulization of the liposomal amikacinformulation aerosolized with twenty four nebulizer aerosol heads with 8mL reservoir handsets were measured. The liposome size ranged from 248.9nm to 288.6 nm, with an average of 264.8±6.7 nm (n=72). These resultsare provided in Table 8. The pre-nebulization liposome mean diameter wasapproximately 285 nm (284.5 nm±6.3 nm).

TABLE 8 Liposome size post-nebulization Aerosol Head Mean Diameter Run #(nm) 1 1 270.9 2 2 274.6 3 3 253.9 4 4 256.3 5 5 274.0 6 6 273.6 7 7260.0 8 8 268.1 9 9 264.7 10 10 254.8 11 11 266.9 12 12 270.0 13 1 269.614 2 271.2 15 3 254.6 16 4 270.7 17 5 260.8 18 6 252.3 19 7 267.8 20 8265.0 21 9 261.5 22 10 258.0 23 11 248.9 24 12 262.4 25 1 266.0 26 2270.4 27 3 268.6 28 4 266.6 29 5 259.4 30 6 265.2 31 7 262.4 32 8 257.733 9 264.1 34 10 258.5 35 11 273.4 36 12 260.2 37 13 266.0 38 14 270.239 15 268.2 40 16 266.2 41 17 265.5 42 18 268.5 43 19 263.3 44 20 257.845 21 271.3 46 22 266.2 47 23 270.6 48 24 269.7 49 13 269.1 50 14 265.751 15 258.7 52 16 268.0 53 17 266.2 54 18 254.0 55 19 263.9 56 20 265.357 21 264.5 58 22 266.5 59 23 264.8 60 24 271.7 61 13 259.8 62 14 268.863 15 265.9 64 16 274.7 65 17 256.2 66 18 269.7 67 19 257.7 68 20 255.769 21 264.8 70 22 288.6 71 23 252.1 72 24 263.4 Average 264.8 ± 6.7

All, documents, patents, patent applications, publications, productdescriptions, and protocols which are cited throughout this applicationare incorporated herein by reference in their entireties for allpurposes.

The embodiments illustrated and discussed in this specification areintended only to teach those skilled in the art the best way known tothe inventors to make and use the invention. Modifications and variationof the above-described embodiments of the invention are possible withoutdeparting from the invention, as appreciated by those skilled in the artin light of the above teachings. It is therefore understood that, withinthe scope of the claims and their equivalents, the invention may bepracticed otherwise than as specifically described.

1. A system for treating or providing prophylaxis against a pulmonaryinfection in a patient, comprising: (a) a pharmaceutical formulationcomprising a liposomal complexed aminoglycoside, wherein the formulationis an aqueous dispersion, and the lipid component of the liposomeconsists of electrically neutral lipids, and (b) a nebulizer whichgenerates an aerosol of the pharmaceutical formulation at a rate greaterthan about 0.53 g per minute, wherein the mass median aerodynamicdiameter (MMAD) of the aerosol is less than about 4.2 μm, as measured bythe Anderson Cascade Impactor (ACI), or less than about 4.9 μm, asmeasured by the Next Generation Impactor (NGI). 2.-146. (canceled)